Revision as of 16:52, 29 June 2016

Labjournal

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Samples

Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of Phytochrome B for protein fusion.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

2 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 1 ml LB-medium to each tube.

Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

100 µl of the cell suspension was plated on one chloramphenicol plate.

The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Investigator: Jeff, Rosario

Aim of the experiment: Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Sequencing of RFP-Generator (RFC25, pSB1C3)

Investigator: Jeff, Rosario

Aim of the experiment: Sequencing of RFP-Generator (RFC25, pSB1C3)

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer)

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Procedure:

pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.

Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).

4 colonies were picked.

These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).

Procedure:

Batch for analytical digestion for P4 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P4
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P5 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P5
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	P4	P5
	Mutation successful	Mutation successful!

Parts are compliant and do not contain RFC25 forbidden restriction sites.

500px

Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Investigator: Jeff, Rosario, Florian

Aim of the experiment: Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different vectors we sequenced received the following barcodes:

- ADH in pTUM100: FR01002265

- TEF1 in pTUM100: FR01002266

- TEF2 in pTUM100: FR01002266

- GAL in pTUM100: FR01002268

Sequencing of TEF2 in pTUM100 was not interpretable. The other sequences were consistent with the sequences in the parts registry.

Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Florian

Aim of the experiment: Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10

Investigator: Jeff, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.

Procedure:

Batch for analytical digestion for P7 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P7
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P8 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P8
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P9 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P9
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Batch for analytical digestion for P10 with NgoMIV+AgeI-HF

volume	reagent
2.5 µl	Plasmid DNA P10
2 µl	NEBuffer 4 (10x)
0.25 µl	NgoMIV (10 U/µl)
0.25 µl	AgeI-HF (20 U/µl)
15 µl	ddH2O
=20 µl	TOTAL

Incubation for 90 min at 37 °C.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder	P7	P8	P9	P10
	Part is correct	Part is correct	Part is correct	Part is correct

500px

Transformation of E. coli XL1 blue with

Investigator:

Aim of the experiment: Transformation of E. coli XL1 blue.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of µl LB-medium to each tube.

The cell suspension was plated on ampicillin plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.

Week 1, May 16th - May 22nd

Monday, May 16th

Streptavidin plasmids control

Investigator: JB, LK, JH

Aim of the experiment: Verification of cloning

Procedure:

MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen) (4 clones each of pSA1, pSAm1 in pASK75)
analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF), 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O
5 µl on 1% agarose gel electrophoresis of digestion

Results: successful cloning verified, stored at -20 °C

1. Lane: 5 µl Thermo Fisher, 1kb Ladder

2. to 9. Lane: 5 µl digestions of P6 to P13, band of SA (mut1) at about 300 bp, band of digested plasmid at about 3.000 bp

Streptavidin expression_trafo BL21

Investigator: JB, JH

Aim of the experiment: expression of pSA1 and pSAm1 in E. Coli BL21

Procedure:

transformation according to protocol of P6 and P10 in competent E. Coli BL21

result: plates (LB Amp) in incubator for further processing (37 °C)

Tuesday, May 17th

SDS Gel Analysis

Investigator: CG

Aim of the experiment: SDS gel analysis of collagen 1/2, eGFP, fraction 30 of egg-precipitation

Procedure:

mixing of 80 µl samples with 20 µl SDS buffer and heating at 95°C for 10 min. 1 d staining, 1 d unstaining

Results: successful cloning verified, stored at -20 °C

1. Lane: 8 µl Marker (Thermo Fisher #26610)

2. Lane: fraction 30 (IEC), 3 µl, band at 35 kDa, Avidin expected at 16 kDa

3. Lane: eGFP, 12 µl, band at 27 kDa eGFP expected at 27 kDa, many impurities

4. Lane: Collagen 1, 12 µl, no sharp band

5. Lane: Collagen 1, 12 µl, no sharp band

Minipreps pSb1C3-AviTag, -A3C5, pASK75-(SA1), -(SAm1)

Investigator: CR, CG

Aim of the experiment: Verification of cloning

Procedure:

MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF) for pASK plasmids and 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O - 5 µl on 1% agarose gel electrophoresis of digestion

result: : successful cloning verified for pASK plasmids, repetition of pSb1C3 plasmids, stored at -20 °C

1. Lane: 5 µl Thermo Fisher, 1 kb Ladder

2. Lane: 5 µl digestion of pSb1C3-AviTag

3. Lane: 5 µl digestion of pSb1C3-A3C5

5. Lane: 5 µl digestion of pASK75(SA1), EB elution

6. Lane: 5 µl digestion of pASK75(SAmut1), EB elution

7. Lane: 5 µl digestion of pASK75(SAmut1), H2O elution

Inoculation of pre-culture with BL21 (pASK75 (SA1)) in LB-medium

Investigator: CR

Aim of the experiment: Preculture for streptavidin expression in TB-medium

Procedure:

Add 50 µL ampicillin in 50 mL LB-medium

Picking colonies from BL21 (pASK75 (SA1))

Inoculate LB-medium

Incubate at 30°C over night

Wednesday, May 18th

Repetition of analytical gel of pSb1C3-AviTag, -A3C5

Investigator: CG, CR

Aim of the experiment: Verification of cloning

Procedure:

analytic digestion with: 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 8,5 µl Plasmid-DNA
10 µl on 2% agarose gel electrophoresis of digestion

Results:

Inoculation of BL21 (pASK75 (SA1)) culture in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline

Investigator: CR

Aim of the experiment: Production of streptavidin

Procedure:

Ampicillin (2 mL) was added to the Medium (1:1000)
The pre-culture (50 mL) was poured into the Medium
Culture was incubated at 37°C and 140 rpm until OD550 reached 0.5
To induce streptavidin expression anhydro-tetrazycline (200 µL) was added to the culture (1:10000)
The culture was incubated at 37°C and 140 rpm for 4 hours

Results:

Streptavidin expression by BL21

Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium

Investigator: CR, JB, JH

Aim of the experiment: Recombinant expression and purification of Streptavidin

Procedure:

After expression, cultures were transferred into centrifuge tubes and spun down in the centrifuge (4°C, 5000 rpm, 20 mins, F 4X1L rotor)
The supernatant was cast away and the pellet was transferred into a beaker of sufficient size and resuspended in fridge-cooled Tris Buffer B (50 mM Tris/HCl (pH = 8.0), 1 mM EDTA)
The solution was homogenized in the PANDA (ask supervisor)
The resulting lysate was transferred into centrifuge tubes and spun down (4°C, 18,000 rpm, 10 mins, XX34-rotor). The supernatant was cast away and the pellet was resuspended in 6M Gua-HCl (pH = 1.5) at 4°C overnight.

Dialysis of eGFP

Investigator: NA, JH, CR

Aim of the experiment: Purification of eGFP

Procedure:

eGFP was thawed on ice
eGFP was then poured in a dialysis hose (cut-off 14 kDa)
The hose was then placed in ice cold Tris/HCl 20 mM pH 8.0
The dialysis took place at 4°C over nightXX34-rotor). The supernatant was cast away and the pellet was

MiniPrep of quickchanged pNGAL146-A2

Investigator: NA

Aim of the experiment: Extraction of pNGAL146-A2 plasmid from XL1 blue

Procedure:

MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

Sequencing of P14, P15 & P19

Investigator: CR, NA

Aim of the experiment: Sequencing of P14, P15 & P19

Procedure:

Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)
The different plasmids we prepared received the following barcodes:
P14 : FR11326653
P15 : FR11326655
P19 (K4): FR11326654
P16 (K1): FR11326652
P17 (K2): FR11326651
P18 (K3): FR11326650

Digestion of P16, P17, P18 & P19 with AgeI & HindIII + analytical gel

Investigator: NA, JH, CR

Aim of the experiment: Verification of success of quickchange

Procedure:

analytic digestion with: 0,25 µl HindIII (HF), 0,25 µl AgeI (HF), 1 µl SmartCut Buffer, 500 ng plasmid-DNA, fill up with ddH2O (Vtotal= 10µL)
10 µl on 2% agarose gel for electrophoresis

Results: No signal at 600 bp --> quickchange seems to be successful (waiting for sequencing)

Thursday, May 19th

Re-Sequencing of P19

Investigator: CR

Aim of the experiment: Re-Sequencing of P19

Procedure:Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)

The different plasmids we prepared received the following barcodes:

P19 (K4): FR11326649

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CG

Aim of the experiment: Re-Trafo of pSB1C3 RFP for later on: digestion, dephosphorylation and cloning

Procedure: transformation according to protocol of P4 E. Coli XL1

Result: plates (LB Cam) in incubator for further processing (37 °C)

Streptavidin refolding

Investigator: JB

Aim of the experiment: Refolding of denaturated Streptavidin

Procedure: After the pellet had almost completely dissolved in 6M GdmCl, the solution was spun down (4°C, 20 mins, 18,000 rpm). The supernatant was transferred carefully into a falcon tube and the pellet was cast away. Via a hydraulic pump (flow rate: 2x10 ml/min) the lysate was transferred Into 5L PBS 1x. Afterwards the pump was cleaned with technical isopropanol and ELGA water. The solution was stirred overnight at 4°C for refolding.

Biotinylation of BSA

Investigator: JB

Aim of the experiment: Biotinylation of BSA

Procedure: A 100 µM (=6.8 mg/ml) solution of BSA (Albumin fraction V, pH=7, in the fridge in the central lab) was created (V=10 ml). 220 µl of a 100 mM Biotin stock were added. The mixture was stored overnight Iin the fridge (4°C).

Result: Hopefully biotinylated BSA mixture in the fridge (4°C).

Week 2 (May 23rd - May 29th

Week 3 (May 30th - June 5th

Week 4 (June 6th - June 12th)

Week 5 (June 13th - June 19th)

Week 6 (June 20th - June 26th)

Thursday, June 23rd

Miniprep of E. coli Xl1-Blue transformed with ligation product P80/81 (mRuby3 K1/2), P82/83 (EspP K1/2), P84/85 (StrepTag K1/2) and Trafo of K157001

Investigator: Jan, Julian

Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation product F50(K1,2), F51(K1,2), F52(K1,2) and Trafo of K157001 Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Concentrations:

Plasmid	c [ng/µl]
P80	432,7
P81	294,8
P82	450,5
P83	479,0
P84	108,0
P85	356,0
P86	47,2

Friday, June 24th

Analytical digestion and gelelectrophoresis of P83 (EspP K2) and P83 (StrepTag K2)

Investigator: Julian, Niklas, Luisa

Aim of the experiment: Analytical digestion and gelelectrophoresis of P82/83 (EspP K1/2) and P84/85 (StrepTag K1/2).

Procedure:

Batch for analytical digestion for P82-P85 with EcoRI-HF

volume	reagent
0.5/1.0 µl	Plasmid DNA (-/P84)
1 µl	CutSmart buffer (10x)
0.5 µl	EcoRI-HF(10 U/µl)
8/7.5 µl	ddH2O (-/P84)
=10 µl	TOTAL

Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)

Investigator: Julian

Aim of the experiment: Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer). Sequencing primer VF2 was used

The different vectors we sequenced received the following barcodes:

mRuby3 in pSB1C3 (P80): FR11326590

EspP in pSB1C3 (P83): FR11326588

Streptag in pSB1C3 (P85): FR11326587

Transformation of E. coli XL1 blue with F64 (quickchanged P3(pSAm1))

Investigator: Niklas

Aim of the experiment: Transformation of E. coli XL1 blue.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 750 µl LB-medium to each tube.

The cell suspension was plated on ampicillin plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.

--> Quickchange did not work, do again, than new transformation!

Gelextraction of F67(BirA), F68(mRuby), F69(EGFR-TMD), F70(pSB1C3) and F71(pSB1C3)

Investigator: Niklas

Aim of the experiment: Gelextraction of F67(BirA(Digest. F59 [EcoRI; SpeI])), F68(mRuby(Digest. F60 [NgoMIV; SpeI])), F69(EGFR-TMD(Digest. F60 [NgoMIV; SpeI]), F70(pSB1C3(digest. P74 [NgoMIV; SpeI]) and F71(pSB1C3(digest. P74 [EcoRI; SpeI])

Procedure:

Gelextraction was performed by manufacturers protocol (Qiagen).

Saturday, June 25th

Miniprep of E. coli Xl1-Blue transformed with P60 (mRuby/EGFR),F58 (Ligation pASK75 + Streptactin), F65 (CMV + CD4), F66 (CMV + EGFR), P70 (Short Linker)

Investigator: Niklas

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Concentrations:

Plasmid	c [ng/µl]
P87	81
P88	34,5
P89	86,3
P90	108,5
P91	417,4

Analytical digestion and gelelectrophoresis of F64 (quickchanged P3) for verification of succesful quickchange

Investigator: Niklas

Procedure:

Analytical digestion with NdeI and gelelectrophoresis. If quickchange worked there should be a band at about 3200 bp (only one restriction site left)

Incubation over night at room temperature.

Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

Just a band showing a few bp (Primer), there is no plasmid band -> Quickchange did not work

Inoculation of colonies from Ligation of F69 + F70 (EGFR-TMD in pSB1C3)and F44 + F30 (mRuby in pSB1C3)

Investigator: Niklas

Procedure:

6x 4 ml LB+Cam media

Each culture was inoculated with one colony

Incubation at 37°C overnight

Sunday, June 26th

Miniprep of E. coli Xl1-Blue transformed with ligation product F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3)

Investigator: Luisa

Aim of the experiment: Extracting F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3) from E.coli XL-1-blue

Procedure:

Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Concentrations:

Plasmid	c [ng/µl]
P92	162,9
P93	447,9

Repetition of Quick-Change PCR of P3 (pASK + SAm1)

Investigator: Luisa

Procedure:

The QC-PCR was performed according the SOP.

Reaction Mix:

volume	reagent
1,25 µl	Primer O21
1,25 µl	Primer O22
1 µl	dNTP-mix
5 µl	Pfu-Ultra-II reaction buffer
1 µl	template DNA (1:10 dilution of p3)
0,5 µl	Pfu-Ultra-II Polymerase
40,5 µl	ddH2O

digestion of PCR-Product with DpnI for 1h at 37°C

now labeled P94

Transformation of E. coli XL1 blue with P94 (quickchanged P3(pSAm1))

Investigator: Luisa

Aim of the experiment: Transformation of E. coli XL1 blue.

Procedure:

CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

10 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 950 µl LB-medium to each tube.

The cell suspension was plated on ampicillin plates (inclusive rescue plate) for pASK (F72) and on chloramphenicol plates for P92 and P93 and incubated over night at 37 °C in the incubator.

Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75)

Investigator: Luisa

Aim of the experiment: Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75).

Procedure:

Batch for analytical digestion for P92 and P93 with EcoRI-HF and PstI-HF

volume	reagent
1 µl	Plasmid DNA
1 µl	CutSmart buffer (10x)
0.5 µl	EcoRI-HF(10 U/µl) and PstI-HF (10 U/µl) for P92/93, NdeI (10 U/µl) for P94
7.5/7 µl	ddH2O
=10 µl	TOTAL

Results:

1. band (P92): no mRuby at 700bp visible, only empty vector
2. band (P93): empty vector and EGFR --> perfect
3. band (P94): no signal at all --> repetition of QC-PCR

Week 7 (June 27th - July 3rd)

Monday, June 27th

Sequencing of P67 (EGFR-Signalpeptid)

Investigator: Niklas

Procedure:

Sequencing batch was prepared after manufacturer's protocol. (15 µl of plasmid DNA (100 ng) and 2 µl sequencing primer (VF2))

FR11326586

Repetition of Quick-Change PCR of P3 (pASK + SAm1)

Investigator: Luisa

Procedure:

The QC-PCR was performed according the SOP.

Reaction Mix:

volume	reagent
1,25 µl	Primer O21
1,25 µl	Primer O22
1 µl	dNTP-mix
5 µl	Pfu-Ultra-II reaction buffer
1 µl	template DNA (1:10 dilution of p3)
0,5 µl	Pfu-Ultra-II Polymerase
40,5 µl	ddH2O

Digestion of PCR-Product with DpnI for 1h at 37°C.

Transformation of 10µl into component E.coli XL-1-blue, according to SOP (1h incubation at 37°C necessary despite AmpR).

PCR of Genesynthesis 3 and 4

Investigator: Luisa

Aim of Experiment: Amplification of Genesynthesis 3 (contains BAP and IGKappa) and 4 (contains A3C5-tag and BM40)

Procedure:

The PCR was performed according the SOP.

Reaction Mix:

volume	reagent
2,5 µl	Primer VF2
2,5 µl	Primer VR2
1 µl	dNTP-mix
10 µl	Q5 Polymerase reaction buffer
1 µl	template DNA (1:10 dilution of p3)
0,5 µl	Q5-Polymerase
18 µl	ddH2O

Setup: iGEM_standard (Promega-cycler)

temperature	time
98°C	2min
98°C	10sec
66°C	30sec
72°C	30sec
72°C	2min
4°C	hold

the batches were then purified using the Quiagen PCR-Purification Kit.

Analytical digestion and gelelectrophoresis of P88 , P89 and P90

Investigator: Niklas

Aim of experiment: Analytical digestion and gelelectrophoresis of P88 (pASK75 + Streptactin, former F58), P89 (CMV + CD4, former F65) and P90 (CMV + EGFR-signal-peptide, former F66)

Procedure:

Batches for analytical digestions:

P88: EcoRI

P89: EcoRI and PstI

P90: EcoRI

volume	reagent
5,8/2,3/1,8 µl	Plasmid DNA (P88/P89/P90)
1 µl	CutSmart buffer (10x)
0.5 µl	EcoRI-HF(10 U/µl)/ PstI
required amount for total volume of 10 µl	ddH2O

Ligation of F67 and F71, Transformation of E. coli XL1 blue afterwards

Investigator: Niklas

Aim of the experiment: Ligation of F67 (BirA) and F71 (empty pSB1C3), Transformation of E. coli XL1 blue afterwards.

Procedure:

volume	reagent
2,4 µl	Vektor
7,6 µl	Insert
2 µl	10X DNA-Ligase-buffer
1 µl	T4-Ligase
7 µl	ddH₂O
=20 µl	TOTAL

CaCl2 competent E. coli XL1-Blue cells were taken out of stock in -80 °C freezer and were gently thawed on ice.

7 µl of DNA was added to 100 µl of competent cells and gently mixed.

30 min incubation on ice

5 min. heat shock at 37 °C

Adding of 750 µl LB-medium to each tube.

Incubation for 1 hour at 37 °C

The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C in the cell-culture shaker.

next step: analytic digestion of transformation was successful

Digestion of PCR on genesynthesis 3 and 4, and pSB1C3

Investigator: Luisa

Aim of experiment: Division of Leptin, IGKappa, A3C5, BM40 and BAP using SapI, HindIII, XbaI, AgeI for both batches.

Procedure:

Batches for analytical digestions:

volume	reagent
1 µl each	enzyme (SapI, HindIII, XbaI, AgeI)
5 µl	CutSmart buffer (10x)
41 µl	DNA (purified PCR-products of GSY3 and 4)

Additionally 10µg of the vector P74 was digested with XbaI and AgeI in 100µl batch (2µl of each enzyme, 10µl of Cut-Smart buffer). Digestion was performed over night and purified via gelelectrophoresis and gelextraction according to the manufacturer's protocoll. --> Now labeled F80.

Analytical digestion and gelelectrophoresis of P80 , P78 and P85

Investigator: Julian

Aim of experiment: Analytical digestion and gelelectrophoresis of P80 (mRuby3), P78 (NanoLuc) and P85(Strep-Tag)

Procedure:

Batches for analytical digestions:

P80 and P78: EcoRI and AgeI

P85: EcoRI and NgoMIV

volume	reagent
10 µl	Plasmid DNA
32 µl	ddH2O
5 µl	CutSmart buffer (10x)
1.5 µl	each enzyme(10 U/µl)/ PstI
50 µl	TOTAL

File:Muc16 .JPG

Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3 with Strep-Tag) and Transformation into E. coli XL1 blue

Investigator: Luisa

Aim of the experiment: Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3+Strep-Tag), Transformation of E. coli XL1 blue afterwards.

Procedure:

volume	reagent
4,3µl(for F75), 8,2µl (for F76)	Vector
12,7µl (F75), 8,8 (F76)	Insert
2 µl	10X DNA-Ligase-buffer
1 µl	T4-Ligase
=20 µl	TOTAL

Ligation was incubated at RT for 1,5h.

CaCl2 competent E. coli XL1-Blue cells were taken out of stock in -80 °C freezer and were gently thawed on ice.

7 µl of DNA was added to 100 µl of competent cells and gently mixed.

15 min incubation on ice

5 min. heat shock at 37 °C

Adding of 950 µl LB-medium to each tube.

Incubation for 1 hour at 37 °C

The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C in the incubator.

Chemical biotinylation of BSA

Investigator: Niklas

Procedure:

BSA was chemically biotinylated with a 20x and 40x molar excess:

10 ml of 100 mM borate buffer with 50 mM NaCl (pH 8.85)

dissolve BSA (10 mg/ml)

Add biotin-NHS-ester: 20,5 mg for 40x molar excess

reaction over night

</div>

Tuesday, June 28th

</div>

Wednesday, June 29th

</div>

@@ Line 1: / Line 1: @@
-{{LMU-TUM_Munich}}
+== Labjournal ==
-{{LMU-TUM_Munich/16style}}
-{{Team:TU-Munich/ExCol}}
-{{Team:TU-Munich/LabHeader}}
+<html>
+        <form id="labselect"><fieldset class="ui-widget ui-widget-content ui-corner-left">
+        <b>Display:</b><br>
+        <input class="labcheckbox" type="checkbox" name="category" value="general" id="ui-test7" /><b style="color: rgb(115, 208, 255);">General</b><br>
+        <input class="labcheckbox" type="checkbox" name="category" value="streptavidin" id="ui-test1" /><b style="color: rgb(166, 126, 166);">Streptavidin</b><br>
+        <input class="labcheckbox" type="checkbox" name="category" value="linkers" id="ui-test2" /><b style="color: rgb(116, 183, 112);">Linkers</b><br>
+        <input class="labcheckbox" type="checkbox" name="category" value="receptor" id="ui-test3" /><b style="color: rgb(255, 122, 97);">Receptor</b><br>
+        <input class="labcheckbox" type="checkbox" name="category" value="optogenetics" id="ui-test5" /><b style="color: rgb(0, 32, 96);">Optogenetics</b><br>
+        <a href="#" id="ExAll">Expand All ...</a><br>
+        <a href="#" id="ColAll">Collapse All ...</a><br>
-<div id="wikicontent-container">
+        <b>Jump to:</b><br>
-<div id="wikicontent">
+        <a href="#Week_1">Week 1</a> 02.05-08.05<br>
-<div style="text-align:center;"><span style="color:#6fac47;">'''Monday, May 16th'''</span> </div>
+        <a href="#Week_2">Week 2</a> 09.05-15.05<br>
+        <a href="#Week_3">Week 3</a> 16.05-22.05<br>
+        <a href="#Week_4">Week 4</a> 23.05-29.05<br>
+        <a href="#Week_5">Week 5</a> 30.05-05.06<br>
+        <a href="#Week_6">Week 6</a> 06.06-12.06<br>
+        <a href="#Week_7">Week 7</a> 13.06-19.06<br>
+        </fieldset>
+        </form>
+        <div id="ladder" class="ui-widget ui-widget-content ui-corner-right">
+        <br><b>1 kbp GeneRuler:</b><br><br>
+        <img src="https://static.igem.org/mediawiki/2012/e/e3/TUM12_1000bp.jpg"></img><br>
+        <br><b>100 bp GeneRuler:</b><br><br>
+        <img src="https://static.igem.org/mediawiki/2012/d/da/TUM12_100bp.jpg"></img><br>
+        <br><b>PageRuler Plus:</b><br><br>
+        <img src="https://static.igem.org/mediawiki/2012/8/8c/TUM12_250kDa.jpg"></img><br>
+        </div>
+</html>
-<div style="color:#2e74b5;"></div>
+<div class="labbook">
+</div>
-<span style="background-color:#ffff00;">'''Streptavidin plasmids_control'''</span><span style="background-color:#ffff00;"> </span>
+=Samples=
+<div class="week" id="WWeek_0">
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">JB, LK, JH</span>
+<div class="safety_mechanism">
+===  Transformation of ''E.&nbsp;coli'' XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3) ===
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Verification of cloning</span>
+'''Investigator: Jeff, Rosario'''
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen) (4 clones each of pSA1, pSAm1 in pASK75) </span>
+'''Aim of the experiment:''' Transformation of Phytochrome B for protein fusion.
-* <span style="background-color:#ffff00;">analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF), 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O</span>
-* <span style="background-color:#ffff00;">5 µl on 1% agarose gel electrophoresis of digestion</span>
+'''Procedure:'''
+* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
-<span style="background-color:#ffff00;">'''Result: '''</span><span style="background-color:#ffff00;">successful cloning verified, stored at -20 °C</span>* <span style="background-color:#ffff00;">1. Lane: 5 µl Thermo Fisher, 1kb Ladder</span>
+* 2&nbsp;µl of DNA was added to 100&nbsp;µl of competent cells and gently mixed.
-* <span style="background-color:#ffff00;">2. to 9. Lane: 5 µl digestions of P6 to P13, band of SA (mut1) at about 300 bp, band of digested plasmid at about 3.000 bp</span>
+* 30&nbsp;min incubation on ice
+* 5&nbsp;min. heat shock at 37&nbsp;°C
-[[Image:|top]]
+* Adding of 1&nbsp;ml LB-medium to each tube.
+* Incubation for 45&nbsp;min at 37&nbsp;°C in the 180&nbsp;rpm cell-culture shaker.
+* 100&nbsp;µl of the cell suspension was plated on one chloramphenicol plate.
+* The rest were centrifuged for 1&nbsp;min at 13000&nbsp;rpm and the supernatant was dicarded.
-<span style="background-color:#ffff00;color:#2e74b5;">'''Streptavidin expression_trafo BL21'''</span>
+* The pellet was resuspended in 100&nbsp;µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
+</div>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">JB, JH </span>
+<div class="general">
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> expression of pSA1 and pSAm1 in E. Coli BL21</span>
+===  Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator ===
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span><span style="background-color:#ffff00;">transformation according to protocol of P6 and P10 in competent E. Coli BL21 </span>
+'''Investigator: Jeff, Rosario'''
-<span style="background-color:#ffff00;">'''Result: '''</span><span style="background-color:#ffff00;">plates (LB Amp) in incubator for further processing (37 °C)</span>
+'''Aim of the experiment:''' Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator
+'''Procedure:'''
+* Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
+</div>
+<div class="general">
-<div style="text-align:center;"><span style="color:#6fac47;">'''Tuesday, May 17th'''</span> </div>
+=== Sequencing of RFP-Generator (RFC25, pSB1C3) ===
+'''Investigator: Jeff, Rosario'''
-<span style="background-color:#ffff00;color:#2e74b5;">'''SDS Gel Analysis'''</span>
+'''Aim of the experiment:''' Sequencing of RFP-Generator (RFC25, pSB1C3)
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">CG</span>
+'''Procedure:'''
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> SDS gel analysis of collagen 1/2, eGFP, fraction 30 of egg-precipitation</span>
+Sequencing batch were prepared after manufacturer's protocol. (15&nbsp;µl of plasmid DNA (50 - 100&nbsp;ng) and 2&nbsp;µl sequencing primer)
+</div>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span><span style="background-color:#ffff00;">mixing of 80 µl samples with 20 µl SDS buffer and heating at 95°C for 10 min. 1 d staining, 1 d unstaining</span>
+<div class="safety_mechanism">
-<span style="background-color:#ffff00;">'''Result:'''</span>* <span style="background-color:#ffff00;">1. Lane: 8 µl Marker (Thermo Fisher #26610)</span>
+=== Picking of of ''E.&nbsp;coli'' XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3) ===
-* <span style="background-color:#ffff00;">2. Lane: fraction 30 (IEC), 3 µl, band at 35 kDa, Avidin expected at 16 kDa</span>
-* <span style="background-color:#ffff00;">3. Lane: eGFP, 12 µl, band at 27 kDa eGFP expected at 27 kDa, many impurities</span>
-* <span style="background-color:#ffff00;">4. Lane: Collagen 1, 12 µl, no sharp band</span>
-* <span style="background-color:#ffff00;">5. Lane: Collagen 1, 12 µl, no sharp band</span>
+'''Investigator: Jeff, Rosario, Florian'''
+'''Aim of the experiment:''' Picking of of ''E.&nbsp;coli'' XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
+'''Procedure:'''
+* pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.
+* Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4&nbsp;mL of LB-medium + 4&nbsp;µL chloramphenicol(1000x).
+* 4 colonies were picked.
-[[Image:|top]]
+* These tubes were transferred in a cell culture shaker at 37&nbsp;°C and were incubated overnight
+</div>
+<div class="general">
+=== Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5) ===
-<span style="background-color:#ffff00;color:#2e74b5;">'''Minipreps pSb1C3-AviTag, -A3C5, pASK75-(SA1), -(SAm1)'''</span>
+'''Investigator: Jeff, Rosario, Florian'''
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">CR, CG</span>
+'''Aim of the experiment:''' Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Verification of cloning</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)</span>
+'''Procedure:'''
+* Batch for analytical digestion for P4 with NgoMIV+AgeI-HF
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|2.5&nbsp;µl
+|Plasmid DNA P4
+|-
+|2&nbsp;µl
+|NEBuffer 4 (10x)
+|-
+|0.25&nbsp;µl
+|NgoMIV (10&nbsp;U/µl)
+|-
+|0.25&nbsp;µl
+|AgeI-HF (20&nbsp;U/µl)
+|-
+|15&nbsp;µl
+|ddH2O
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
+|}
-* <span style="background-color:#ffff00;">analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF) for pASK plasmids and 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O</span>
+* Batch for analytical digestion for P5 with NgoMIV+AgeI-HF
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
-<div style="margin-left:0.4917in;margin-right:0in;"><span style="background-color:#ffff00;">- 5 µl on 1% agarose gel electrophoresis of digestion</span></div>
+|-
+|2.5&nbsp;µl
+|Plasmid DNA P5
-<span style="background-color:#ffff00;">'''Result: '''</span><span style="background-color:#ffff00;">successful cloning verified for pASK plasmids, repetition of pSb1C3 plasmids, stored at -20 °C</span>* <span style="background-color:#ffff00;">1. Lane: 5 µl Thermo Fisher, 1 kb Ladder</span>
+|-
-* <span style="background-color:#ffff00;">2. Lane: 5 µl digestion of pSb1C3-AviTag</span>
+|2&nbsp;µl
-* <span style="background-color:#ffff00;">3. Lane: 5 µl digestion of pSb1C3-A3C5</span>
+|NEBuffer 4 (10x)
-* <span style="background-color:#ffff00;">4. Lane: 5 µl digestion of pASK75(SA1), EB elution</span>
+|-
-* <span style="background-color:#ffff00;">5. Lane: 5 µl digestion of pASK75(SA1), H2O elution</span>
+|0.25&nbsp;µl
-* <span style="background-color:#ffff00;">6. Lane: 5 µl digestion of pASK75(SAmut1), EB elution</span>
+|NgoMIV (10&nbsp;U/µl)
-* <span style="background-color:#ffff00;">7. Lane: 5 µl digestion of pASK75(SAmut1), H2O elution</span>
+|-
+|0.25&nbsp;µl
+|AgeI-HF (20&nbsp;U/µl)
+|-
+|15&nbsp;µl
+|ddH2O
-[[Image:|top]]
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
-<div style="color:#2e74b5;"></div>
+|}
-<div style="color:#2e74b5;"></div>
-<div style="color:#2e74b5;"></div>
-<span style="background-color:#ffff00;color:#2e74b5;">'''Inoculation of pre-culture with BL21 (pASK75 (SA1)) in LB-medium '''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">CR</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Preculture for streptavidin expression in TB-medium</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">Add 50 µL ampicillin in 50 mL LB-medium </span>
-* <span style="background-color:#ffff00;">Picking colonies from BL21 (pASK75 (SA1))</span>
-* <span style="background-color:#ffff00;">Inoculate LB-medium</span>
-* <span style="background-color:#ffff00;">Incubate at 30°C over night</span>
-<div style="text-align:center;"><span style="color:#6fac47;">'''Wednesday, May 18th'''</span> </div>
-<span style="background-color:#ffff00;color:#2e74b5;">'''Repetition of analytical gel of pSb1C3-AviTag, -A3C5'''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">CG, CR</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Verification of cloning</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">analytic digestion with: 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 8,5 µl Plasmid-DNA</span>
-* <span style="background-color:#ffff00;">10 µl on 2% agarose gel electrophoresis of digestion</span>
-<span style="background-color:#ffff00;">'''Result:'''</span>
-[[Image:|top]]
-'''Nächstes Mal 1kb ladder!!!!'''
-<span style="background-color:#ffff00;color:#2e74b5;">'''Inoculation of BL21 (pASK75 (SA1)) </span><span style="background-color:#ffff00;color:#2e74b5;">culture</span><span style="background-color:#ffff00;color:#2e74b5;"> in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline'''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">CR</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Production of streptavidin</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">Ampicillin (2 mL) was added to the Medium (1:1000)</span>
-* <span style="background-color:#ffff00;">The pre-culture (50 mL) was poured into the Medium</span>
-* <span style="background-color:#ffff00;">Culture was incubated at 37°C and 140 rpm until OD</span><span style="background-color:#ffff00;"><sub>550</sub></span><span style="background-color:#ffff00;"> reached 0.5</span>
-* <span style="background-color:#ffff00;">To induce streptavidin expression anhydro-tetrazycline (200 µL) was added to the culture (1:10000)</span>
-* <span style="background-color:#ffff00;">The culture was incubated at 37°C and 140 rpm for 4 hours</span>
-<span style="background-color:#ffff00;">'''Result:'''</span>* <span style="background-color:#ffff00;">Streptavidin expression by BL21</span>
-<span style="background-color:#ffff00;color:#2e74b5;">'''Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium '''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">CR, JB, JH </span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Recombinant expression and purification of Streptavidin</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">After expression, cultures were transferred into centrifuge tubes and spun down in the centrifuge (4°C, 5000 rpm, 20 mins, F 4X1L rotor) </span>
-* <span style="background-color:#ffff00;">The supernatant was cast away and the pellet was transferred into a beaker of sufficient size and resuspended in fridge-cooled Tris Buffer B (50 mM Tris/HCl (pH = 8.0), 1 mM EDTA)</span>
-* <span style="background-color:#ffff00;">The solution was homogenized in the PANDA (ask supervisor)</span>
-* <span style="background-color:#ffff00;">The resulting lysate was transferred into centrifuge tubes and spun down (4°C, 18,000 rpm, 10 mins, XX34-rotor). The supernatant was cast away and the pellet was resuspended in 6M Gua-HCl (pH = 1.5) at 4°C overnight.</span>
-<span style="background-color:#ffff00;color:#2e74b5;">'''Dialysis of eGFP'''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">NA, JH, CR</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Purification of eGFP</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">eGFP was thawed on ice</span>
-* <span style="background-color:#ffff00;">eGFP was then poured in a dialysis hose (cut-off 14 kDa)</span>
-* <span style="background-color:#ffff00;">The hose was then placed in ice cold Tris/HCl 20 mM pH 8.0</span>
-* <span style="background-color:#ffff00;">The dialysis took place at 4°C over night</span>
-<div style="margin-left:0.4917in;margin-right:0in;"></div>
-<span style="background-color:#ffff00;color:#2e74b5;">'''MiniPrep of quickchanged pNGAL146-A2'''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">NA</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Extraction of pNGAL146-A2 plasmid from XL1 blue</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)</span>
-<span style="background-color:#ffff00;color:#2e74b5;">'''Sequencing of P14, P15 & P19'''</span>
-<span style="background-color:#ffff00;">'''Investigator:'''</span><span style="background-color:#ffff00;"> CR, NA</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Sequencing of P14, P15 & P19</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>
-<span style="background-color:#ffff00;">Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)</span>
-<span style="background-color:#ffff00;">The different plasmids we prepared received the following barcodes:</span>* <span style="background-color:#ffff00;">P14 : FR11326653</span>
-* <span style="background-color:#ffff00;">P15 : FR11326655</span>
-* <span style="background-color:#ffff00;">P19 (K4): FR11326654</span>
-* <span style="background-color:#ffff00;">P16 (K1): FR11326652</span>
-* <span style="background-color:#ffff00;">P17 (K2): FR11326651</span>
-* <span style="background-color:#ffff00;">P18 (K3): FR11326650</span>
-<div style="color:#2e74b5;"></div>
-<span style="background-color:#ffff00;color:#2e74b5;">'''Digestion of P16, P17, P18 & P19 with AgeI & HindIII + analytical gel'''</span>
-<span style="background-color:#ffff00;">'''Investigator: '''</span><span style="background-color:#ffff00;">NA, JH, CR</span>
-<span style="background-color:#ffff00;">'''Aim of the experiment:'''</span><span style="background-color:#ffff00;"> Verification of success of quickchange</span>
-<span style="background-color:#ffff00;">'''Procedure:'''</span><span style="background-color:#ffff00;"> </span>* <span style="background-color:#ffff00;">analytic digestion with: 0,25 µl HindIII (HF), 0,25 µl AgeI (HF), 1 µl SmartCut Buffer, 500 ng plasmid-DNA, fill up with ddH</span><span style="background-color:#ffff00;"><sub>2</sub></span><span style="background-color:#ffff00;">O (V</span><span style="background-color:#ffff00;"><sub>total</sub></span><span style="background-color:#ffff00;"><nowiki>= 10µL)</nowiki></span>
-* <span style="background-color:#ffff00;">10 µl on 2% agarose gel for electrophoresis </span>
-<span style="background-color:#ffff00;">'''Result:'''</span>
-[[Image:|top]]
-<span style="background-color:#ffff00;">No signal at 600 bp --> quickchange seems to be successful (waiting for sequencing)</span>
-<div style="text-align:center;"><span style="color:#6fac47;">'''Thursday, May 19th'''</span> </div>
-<span style="color:#2e74b5;">'''Re-Sequencing of P19'''</span>
-'''Investigator:''' CR
-'''Aim of the experiment:''' Re-Sequencing of P19
-'''Procedure:'''
-Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)
-The different plasmids we prepared received the following barcodes:* P19 (K4): FR11326649
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
-'''Investigator: '''CG
-'''Aim of the experiment:''' Re-Trafo of pSB1C3 RFP for later on: digestion, dephosphorylation and cloning
-'''Procedure:''' transformation according to protocol of P4 E. Coli XL1
-'''Result: '''plates (LB Cam) in incubator for further processing (37 °C)
-<span style="color:#2e74b5;">'''Streptavidin refolding'''</span>
-'''Investigator: JB'''
-'''Aim of the experiment:''' Refolding of denaturated Streptavidin
-'''Procedure:''' After the pellet had almost completely dissolved in 6M GdmCl, the solution was spun down (4°C, 20 mins, 18,000 rpm). The supernatant was transferred carefully into a falcon tube and the pellet was cast away. Via a hydraulic pump (flow rate: 2x10 ml/min) the lysate was transferred Into 5L PBS 1x. Afterwards the pump was cleaned with technical isopropanol and ELGA water'''.''' The solution was stirred overnight at 4°C for refolding.
-<span style="color:#2e74b5;">'''Biotinylation of BSA'''</span>
-'''Investigator: JB'''
-'''Aim of the experiment:''' Biotinylation of BSA
-'''Procedure: '''A 100 µM (=6.8 mg/ml) solution of BSA (Albumin fraction V, pH=7, in the fridge in the central lab) was created (V=10 ml). 220 µl of a 100 mM Biotin stock were added. The mixture was stored overnight Iin the fridge (4°C).
-'''Result: '''Hopefully biotinylated BSA mixture in the fridge (4°C).
-<div style="text-align:center;"></div>
-<div style="text-align:center;"><span style="color:#6fac47;">'''Friday, May 20</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
-<span style="color:#2e74b5;">'''Qualitative analysis of streptavidin expression</span> '''
-'''Investigator: '''CG
-'''Aim of the experiment:''' SDS gel analysis of recombinant strepatividin expression
-'''Procedure:''' mixing of 80 µl sample with 20 µl SDS buffer and heating at 95°C for 10 min. 1 h staining, 1 night unstaining
-'''Result: '''* 1. Lane: 8 µl Marker (Thermo Fisher #26610)
-* 2. Lane: 12 µl culture aliquot, before induction
-* 3. Lane: 12 µl culture aliquot, after induction
-* 4. Lane: 3 µl culture aliquot of the lysed pelet, Strepatvidin expected at about 16 kDa
-* 5. Lane: 3 µl culture aliquot of the supernatend after lysis, no Strepatvidin expected at about 16 kDa
-* 6. Lane: 1.5 µl culture aliquot of the lysed pelet, Strepatvidin expected at about 16 kDa
-* 7. Lane: 1.5 µl culture aliquot of the supernatend after lysis, no Streptavidin expected at about 16 kDa
-[[Image:|top]]
-<div style="color:#2e74b5;"></div>
-<div style="color:#2e74b5;"></div>
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
-'''Investigator: '''LK
-'''Aim of the experiment:''' <span style="color:#000000;">Inoculation of pre-culture with </span><span style="color:#000000;">''E. coli''</span><span style="color:#000000;"> XL1 (pSb1C3 -RFP) in LB-Chloramphenicol-medium</span>
-'''Procedure:''' * Picking of colonies for <span style="color:#000000;">''E. coli''</span><span style="color:#000000;"> XL1 (pSb1C3 -RFP)</span>
-* Inoculate in 5 ml <span style="color:#000000;">LB-Chloramphenicol-medium</span>
-* Incubate at 37°C over night at 200 rpm
-<span style="color:#2e74b5;">'''Transformation of Biobricks in XL1-blue</span> '''
-'''Investigator: '''NA, JH
-'''Aim of the experiment:''' Transformation
-'''Procedure:'''
--10 µl dd H<sub>2</sub>O in well of interest (standard distribution kit)
--1 µl Plasmid (out of well) to cells
--Transformation according to the SOP
-Used bricks:
-K577893, B0015, R0040, B0032, I14033, K747096
-<span style="color:#4471c4;">'''Ammonium sulfate precipitation of streptavidin'''</span>
-'''Investigator: '''JB
-'''Aim of the experiment:''' Reduction of the protein solution volume and precipitation of streptavidin
-'''Procedure:''' The 5 L protein solution was spun down (20 mins, 5,000 rpm) and the supernatant transferred into a beaker. In order to lower the volume of the solution for ammonium sulfate precipitation, the solution was first filtered via a membrane crossflow pump (membrane: Sartocon 0.45 µm, thick membrane).
-<span style="color:#4472c4;">'''Dialysis of biotinylated BSA'''</span>
-'''Investigator: '''NA
-'''Aim of the experiment:''' purification of biotinylated BSA
-'''Procedure:''' dialysis against Tris/HCl 20 mM, pH 8, 10 mM NaCl over night;
-cut off : 14 kDa
-<div style="text-align:center;"><span style="color:#6fac47;">'''Monday, May 23</span><span style="color:#6fac47;"><sup>rd'''</sup></span></div>
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
-'''Investigator: '''CR
-'''Aim of the experiment:''' Cloning of A3C5 and Avi-Tag into pSB1C3
-'''Procedure:''' * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-* pSB1C3 was digested with AgeI and NgOMIV (10 µL plasmid + 1.5 µL AgeI + 1.5 µL NgoMIV + 3 µL CutSmart + 14 µL ddH<sub>2</sub>O; incubation for 1h, 37°C)
-* FastAP was added and the reaction was incubated for another 2h at 37°C
-* The digestion was purified by gelelctrophoresis (1%, 75V, 1h)
-* Plasmid backbone was cut from the gel
+* Incubation for 90&nbsp;min at 37&nbsp;°C.
+* Analytical gelelectrophoresis was performed at 90&nbsp;V for 60&nbsp;min.
 '''Results:'''
-[[Image:|top]]
+{|cellspacing="0" border="1"
+|1&nbsp;kbp ladder DNA ladder
+|'''P4'''
+|'''P5'''
+|-
+|
+|'''Mutation successful'''
+|'''Mutation successful!'''
+|}
-Signal 1: linearized pSB1C3 (successfull digestion)
+* Parts are compliant and do not contain RFC25 forbidden restriction sites.
-Signal 2: supercoiled pSB1C3 (digestion not successfull)
-Signal 3: RFP-generator
+[[File:TUM13_20130423_RFP_Generator_RFC25_AgeI_NgoMIV.png|500px]]
---> next time gel should run longer (just if you need the back bone)
+</div>
+<div class="general">
-<span style="color:#2e74b5;">'''Filtration of Streptavidin and precipitation with Ammonium-sulfate'''</span>
+=== Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2 ===
-'''Investigator:''' MP, CR
+'''Investigator: Jeff, Rosario, Florian'''
-'''Aim of the experiment:''' Concentration of Streptavidin
+'''Aim of the experiment:''' Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2
-'''Procedure:''' * The solution was filtered via a membrane crossflow pump ('''membrane: Sartocon 0.45 µm, thick membrane).'''
+'''Procedure:'''
-* The final volum was 0.5L
-* The solution was precipitated by addition of Ammonium-sulfate (2 steps: 1. 40% Ammonium-sulfate; 2. 70% Ammonium-sulfate)
-* After the first addition of ammonium-sulfate the solution was stirred (1h, 4°C)
-* Afterwards the solution was centrifuged (10.000 rpm; 30 min) and the supernatant was used for the second precipitation step
-* The solution was stirred again (over night, 4°C)
+Sequencing batch were prepared after manufacturer's protocol. (15&nbsp;µl of plasmid DNA (50 - 100&nbsp;ng) and 2&nbsp;µl sequencing primer).
+The different vectors we sequenced received the following barcodes:
+- ADH in pTUM100:   FR01002265
+- TEF1 in pTUM100:  FR01002266
-<span style="color:#2e74b5;">'''Inoculation of pre-culture with BioBricks in pSB1C3 in LB-Cam'''</span>
+- TEF2 in pTUM100:  FR01002266
-'''Investigator: '''JH, JL
+- GAL in pTUM100:   FR01002268
-'''Aim of the experiment:'''
-'''Procedure:''' * Add 50 µL chloramphenicol in 50 mL LB-medium
+Sequencing of TEF2 in pTUM100 was not interpretable. The other sequences were consistent with the sequences in the parts registry.
-* Picking colonies from K577893; B0015; B0032 (in pSB1C3) *
+</div>
-* Inoculate 4 mL medium (CAM)
-* Incubate at 37°C over night
+<div class="safety_mechanism">
+===  Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)  ===
-<nowiki>*Following cultures didn´t grow and were therefore not inoculated:</nowiki>
+'''Investigator: Jeff, Florian'''
-R0040; K747096; I14033
+'''Aim of the experiment:''' Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).
-<span style="color:#2e74b5;">'''Retransformation of Biobricks in XL1-blue</span> '''
+'''Procedure:'''
-'''Investigator: '''JH, JL, EF
+* Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
-'''Aim of the experiment:''' Transformation
+</div>
-'''Procedure:''' * 10 µl dd H<sub>2</sub>O in well of interest (standard distribution kit)
+<div class="safety_mechanism">
-* 1 µl Plasmid (out of well) to cells
-* Transformation according to the SOP
-* Used bricks:
-* K577893, B0015, R0040, B0032, I14033, K747096
+=== Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10 ===
+'''Investigator: Jeff, Florian'''
+'''Aim of the experiment:''' Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.
+'''Procedure:'''
-<div style="text-align:center;"><span style="color:#6fac47;">'''Tuesday, May 24</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
+* Batch for analytical digestion for P7 with NgoMIV+AgeI-HF
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|2.5&nbsp;µl
+|Plasmid DNA P7
+|-
+|2&nbsp;µl
+|NEBuffer 4 (10x)
+|-
+|0.25&nbsp;µl
+|NgoMIV (10&nbsp;U/µl)
+|-
+|0.25&nbsp;µl
+|AgeI-HF (20&nbsp;U/µl)
+|-
+|15&nbsp;µl
+|ddH2O
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
+|}
+* Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|2.5&nbsp;µl
+|Plasmid DNA P8
+|-
+|2&nbsp;µl
+|NEBuffer 4 (10x)
+|-
+|0.25&nbsp;µl
+|NgoMIV (10&nbsp;U/µl)
+|-
+|0.25&nbsp;µl
+|AgeI-HF (20&nbsp;U/µl)
+|-
+|15&nbsp;µl
+|ddH2O
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
+|}
-<span style="color:#2e74b5;">'''Inoculation of pre-culture with BioBricks in pSB1C3 in LB-Cam'''</span>
+* Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|2.5&nbsp;µl
+|Plasmid DNA P9
+|-
+|2&nbsp;µl
+|NEBuffer 4 (10x)
+|-
+|0.25&nbsp;µl
+|NgoMIV (10&nbsp;U/µl)
+|-
+|0.25&nbsp;µl
+|AgeI-HF (20&nbsp;U/µl)
+|-
+|15&nbsp;µl
+|ddH2O
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
+|}
-'''Investigator:''' CR
+* Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
+{|cellspacing="0" border="1"
-'''Aim of the experiment:'''
+|'''volume'''
+|'''reagent'''
-'''Procedure:''' * Add 50 µL chloramphenicol in 50 mL LB-medium
+|-
-* Picking colonies from K577893; B0015; B0032 (in pSB1C3) *
+|2.5&nbsp;µl
-* Inoculate 4 mL medium (CAM)
+|Plasmid DNA P10
-* Incubate at 37°C over night
+|-
+|2&nbsp;µl
+|NEBuffer 4 (10x)
+|-
+|0.25&nbsp;µl
+|NgoMIV (10&nbsp;U/µl)
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
-'''Investigator:''' CR
-'''Aim of the experiment:''' Gelextraction of Signal 1 from pSB1C3-digestion
-'''Procedure:''' * Gelextraction was performed after manufacturer's protocol (Gelextractionkit, Qiagen)
-<div style="text-align:center;"></div>
-<span style="color:#2e74b5;">'''Ammonium sulfate precipitation'''</span>
-'''Investigator:''' JB
-'''Aim of the experiment:''' Precipitation the 70% saturated ammonium sulfate solution
-'''Procedure:''' The ammonium sulfate solution, which actually was a suspension, was transferred into centrifuge tubes and spun down (10,000 rpm, 45 mins). As it turns out, unfortunately too much ammonium sulfate was used for the precipitation (wrong table). The precipitate was brought back into solution (20 mM Tris/HCl, pH 8.0) and loaded onto an SDS gel for analysis, along with other samples (flowthrough of the filtration from the day before).
-<span style="color:#2e74b5;">'''MiniPrep of BioBricks in pSB1C3 ('''</span><span style="color:#2e74b5;">inoculated on May 23</span><span style="color:#2e74b5;"><sup>th</sup></span><span style="color:#2e74b5;">''')'''</span>
-'''Investigator: '''JH
-'''Aim of the experiment:''' Extraction of BioBricks B0032, B0015 and K577893 in pSB1C3 from XL1 blue
-'''Procedure:''' * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-* concentrations measured (in ng/µL):
-<div style="margin-left:0in;margin-right:0in;">(B0032) 360.2; (B0015) 254.9; (K577893) 99.2</div>
-<span style="color:#2e74b5;">'''Sequencing of P23, P24 & P24'''</span>
-'''Investigator:''' JH
-'''Aim of the experiment:''' Sequencing of P23, P24 & P25 (''x2'')
-'''Procedure:'''
-Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)
-The different plasmids we prepared received the following barcodes:* P23 (VF2) : FR11326647
-* P24 (VF2) : FR11326646
-* P25 (VF2) : FR11326645
-* P25 (VR) : FR11326644
-<div style="text-align:center;"><span style="color:#6fac47;">'''Wednesday, May 25</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
-<span style="color:#2e74b5;">'''Inoculation of pre-culture with tet-repressed GPF (P25) in LB-Cam'''</span>
-'''Investigator: NA'''
-'''Procedure: '''* Add 50 µL chloramphenicol to 50 mL LB-medium
-* Picking colonies from plate (BL21)
-* Incubate over night at 37°C
-'''<nowiki>=> dismissed, because plasmid sequence was inaccurate</nowiki>'''
-<span style="color:#2e74b5;">'''MiniPrep of </span><span style="color:#2e74b5;">K747096, I14033, R0040, B0015, K577893 and B0032</span><span style="color:#2e74b5;"> '''</span>
-'''Investigator:''' CR
-'''Aim of the experiment:''' MiniPrep of BioBricks
-'''Procedure:''' * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-* Concentrations were measured (in ng/µL):
-K747096: 286,2
-I14033: 246,1
-R0040: 243,5
-B0015: 244,1
-K577893: 440,9
-B0032: 259,7
-<span style="color:#2e74b5;">'''PCR of P19 with O3 and O4 '''</span>
-'''Investigator:''' CR
-'''Aim of the experiment:''' Amplification of quickchanged EspP
-'''Procedure:''' *
-* reaction was performed with the Q5 High Fidelity DNA Polymerase (M0491, NEB) and after manufacturer's protocol (PCR Using Q5® High-Fidelity DNA Polymerase, NEB)
-* 274,4 pg Plasmid were used for amplification (1 µL)
-* PCR program (standard):
-{| style="border-spacing:0;width:5.9458in;"
-|- style="border-top:0.5pt solid #bdd6ee;border-bottom:1.5pt solid #9cc2e5;border-left:0.5pt solid #bdd6ee;border-right:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| Step
-|| '''Temperature [°C]'''
-|| Time [s]
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| initial Denaturation
-|| 98
-|| 30
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| 35 cycles
-|| 98
-|| 10
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| 72
-|| 30
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| 72
-|| 60
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| final Extension
-|| 72
-|| 5
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| Hold
-|| 4
-|| ꝏ
 |-
+|0.25&nbsp;µl
+|AgeI-HF (20&nbsp;U/µl)
+|-
+|15&nbsp;µl
+|ddH2O
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
 |}
-* PCR product was purified accoding to manufacturer's protocol (PCR puification kit, Qiagen)
-* DNA was eluted 40 µL EB buffer
+* Incubation for 90&nbsp;min at 37&nbsp;°C.
+* Analytical gelelectrophoresis was performed at 90&nbsp;V for 60&nbsp;min.
+'''Results:'''
+{|cellspacing="0" border="1"
+|1&nbsp;kbp ladder DNA ladder
+|'''P7'''
-<span style="color:#2e74b5;">'''Digestion of purified PCR-product (P19 with O3 and O4)'''</span>
+|'''P8'''
+|'''P9'''
-'''Investigator:''' CR
+|'''P10'''
-'''Aim of the experiment:''' Digestion of EspP with AgeI and NgoMIV to ligate it in pSB1C3 later on
-'''Procedure:''' * Batch for preparative digestion:
-{| style="border-spacing:0;width:5.9458in;"
-|- style="border-top:0.5pt solid #bdd6ee;border-bottom:1.5pt solid #9cc2e5;border-left:0.5pt solid #bdd6ee;border-right:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| '''Volume'''
-|| '''Reagent'''
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| '''38,5 µL'''
-|| PCR product P19 with O3 and O4
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| '''1 µL'''
-|| AgeI
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| '''1 µL'''
-|| NgoMIV
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| '''5 µL'''
-|| CutSmart buffer
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| 4,5
-|| ddH<sub>2</sub>O
-|- style="border:0.5pt solid #bdd6ee;padding-top:0in;padding-bottom:0in;padding-left:0.0785in;padding-right:0.075in;"
-|| '''50 µL'''
-|| '''TOTAL'''
 |-
+|
+|'''Part is correct'''
+|'''Part is correct'''
+|'''Part is correct'''
+|'''Part is correct'''
 |}
-* Incubation for 2 h at 37 °C
+[[File:TUM13_20130424_PhytochromeB_RFC25_AgeI_NgoMIV.png|500px]]
+</div>
+<div class="effectors">
+===  Transformation of ''E.&nbsp;coli'' XL1 blue with  ===
+'''Investigator: '''
+'''Aim of the experiment:''' Transformation of ''E.&nbsp;coli'' XL1 blue.
-<span style="color:#2e74b5;">'''Sequencing of P26, 27, 28, 30'''</span>
-'''Investigator: '''NA
-'''Aim of the experiment:'''
-Sequencing of P26, P27, P28 & P30 (x2)
 '''Procedure:'''
-Sequencing batches were prepared after manufacturer's protocol
+* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
-(15 μL plasmid DNA (50-100 μM) and 2 μL sequencing primer)
+* &nbsp;µl of DNA was added to 100&nbsp;µl of competent cells and gently mixed.
-The different plasmids we prepared received the following barcodes:
+* 30&nbsp;min incubation on ice
-P26 (VF2) : FR11326643
+* 5&nbsp;min. heat shock at 37&nbsp;°C
-P27 (VF2) : FR11326642
+* Adding of &nbsp;µl LB-medium to each tube.
-P28 (VF2) : FR11326641
+* The cell suspension was plated on ampicillin plates (inclusive rescue plate) and incubated over night at 37&nbsp;°C in the cell-culture shaker.
+</div>
-P30 (VF2) : FR11326640
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
-P30 (VR): FR11326639
+=Week 1, May 16th - May 22nd=
+<div class="week" id="WWeek_1">
+=='''Monday, May 16th'''==
+<div class="general">
+=== Streptavidin plasmids control  ===
-<span style="color:#2e74b5;">'''Ion exchange chromatography of eGFP '''</span>
+'''Investigator: JB, LK, JH'''
-'''Investigator: '''NA, JB,JL
+'''Aim of the experiment:''' Verification of cloning
-'''Aim of the experiment: '''purification of eGFP
 '''Procedure:'''
--use Äkta-Purifier (Q-column for eGFP (quarternary ammonium as stationary phase)). Ask Andy before use!
+* MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen) (4 clones each of pSA1, pSAm1 in pASK75)
+* analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF), 1 µl 			SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O
+* 5 µl on 1% agarose gel electrophoresis of digestion
--Pump A: running buffer (20 mM Tris/HCl pH 8); pump B: elution buffer (20 mM Tris/HCl pH 8 ''and'' 1,000 mM (1 M) NaCl
+'''Results:''' successful cloning verified, stored at -20 °C
--basic pumpwash before start
+* 1. Lane: 5 µl Thermo Fisher, 1kb Ladder
--injection in loop
+* 2. to 9. Lane: 5 µl digestions of P6 to P13, band of SA (mut1) at about 300 bp, band of digested plasmid at about 3.000 bp
+</div>
-- gradient of ion strength on column.
+<div class="general">
+=== Streptavidin expression_trafo BL21  ===
+'''Investigator: JB, JH'''
+'''Aim of the experiment:''' expression of pSA1 and pSAm1 in E. Coli BL21
+'''Procedure:'''
+* transformation according to protocol of P6 and P10 in competent E. Coli BL21
+'''result:''' plates (LB Amp) in incubator for further processing (37 °C)
+</div>
+=='''Tuesday, May 17th'''==
+<div class="general">
+=== SDS Gel Analysis  ===
-'''Results:'''
+'''Investigator: CG'''
-Elution of eGFP at ~200 mM NaCl.
+'''Aim of the experiment:''' SDS gel analysis of collagen 1/2, eGFP, fraction 30 of egg-precipitation
-[[Image:|top]]
+'''Procedure:'''
+* mixing of 80 µl samples with 20 µl SDS buffer and heating at 95°C for 10 min. 1 d staining, 1 d unstaining
-<div style="text-align:center;color:#6fac47;"></div>
+'''Results:''' successful cloning verified, stored at -20 °C
-<div style="text-align:center;"><span style="color:#6fac47;">'''Thursday, May 26</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
+* 1. Lane: 8 µl Marker (Thermo Fisher #26610)
+* 2. Lane: fraction 30 (IEC), 3 µl, band at 35 kDa, Avidin expected at 16 kDa
-<span style="color:#2e74b5;">'''Inoculation of cultures with XL-1 F11 and F12 clones'''</span>
+* 3. Lane: eGFP, 12 µl, band at 27 kDa eGFP expected at 27 kDa, many impurities
-'''Investigator: '''JB
+* 4. Lane: Collagen 1, 12 µl, no sharp band
-'''Aim of the experiment:'''
+* 5. Lane: Collagen 1, 12 µl, no sharp band
+</div>
-Inoculation of a 5 ml culture with a clone of the previously transformed F11 (Avi-Tag) F12 (Antibody-binding site).
+<div class="general">
+=== Minipreps pSb1C3-AviTag, -A3C5, pASK75-(SA1), -(SAm1)  ===
+'''Investigator: CR, CG'''
-<span style="color:#2e74b5;">'''Cloning of ligation F14 +F13'''</span>
+'''Aim of the experiment:''' Verification of cloning
-'''Investigator: '''MP, EF, JH
+'''Procedure:'''
-'''Aim of the experiment:''' new biobrick EspP in pSB1C3
+* MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-'''Procedure:''' * F14 was purified by gelelctrophoresis (1%, 90 V, 40 min)
+* analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF) for pASK 	plasmids and 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O - 5 µl on 1% agarose gel electrophoresis of digestion
-* gelextraction was performed after manufacturer's protocol (Gelextractionkit, Qiagen)
-* ligation of F14 (EspP) with F13 (dephos. PSB1C3)
-* transformation according to protocol in competent E.coli XL1 blue
+'''result:''' : successful cloning verified for pASK plasmids, repetition of pSb1C3 plasmids, stored at -20 °C
+* 1. Lane: 5 µl Thermo Fisher, 1 kb Ladder
-'''Results:'''
+* 2. Lane: 5 µl digestion of pSb1C3-AviTag
-[[Image:|top]]
+* 3. Lane: 5 µl digestion of pSb1C3-A3C5
+* 5. Lane: 5 µl digestion of pASK75(SA1), EB elution
-<span style="color:#2e74b5;">'''Transformation of Biobrick B0034 in XL1-blue</span> '''
+* 6. Lane: 5 µl digestion of pASK75(SAmut1), EB elution
-'''Investigator: '''JH
+* 7. Lane: 5 µl digestion of pASK75(SAmut1), H2O elution
+</div>
-'''Aim of the experiment:''' Transformation
+<div class="general">
+=== Inoculation of pre-culture with BL21 (pASK75 (SA1)) in LB-medium   ===
-'''Procedure:'''
+'''Investigator: CR'''
--10 µl ddH<sub>2</sub>O in well of interest (standard distribution kit, plate 4, well 1N)
+'''Aim of the experiment:''' Preculture for streptavidin expression in TB-medium
--1 µl Plasmid (out of well) to cells
+'''Procedure:'''
--Transformation according to the SOP (incl. rescue)
+* Add 50 µL ampicillin in 50 mL LB-medium
+* Picking colonies from BL21 (pASK75 (SA1))
+* Inoculate LB-medium
-<span style="color:#2e74b5;">'''Transformation of Biobricks K577895 and K577894 in XL1-blue</span> '''
+* Incubate at 30°C over night
+</div>
-'''Investigator: '''NA
+=='''Wednesday, May 18th'''==
+<div class="general">
+=== Repetition of analytical gel of pSb1C3-AviTag, -A3C5  ===
-'''Aim of the experiment:''' Transformation
+'''Investigator: CG, CR'''
-'''Procedure:'''
+'''Aim of the experiment:''' Verification of cloning
--10 µl ddH<sub>2</sub>O in well of interest (standard distribution kit, plate 1, wells 14B / 12P)
+'''Procedure:'''
--1 µl Plasmid (out of well) to cells
+* analytic digestion with: 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 		plasmids, 1 µl SmartCut Buffer, 8,5 µl Plasmid-DNA
+* 10 µl on 2% agarose gel electrophoresis of digestion
--Transformation according to the SOP (incl. rescue)
+'''Results:'''
+</div>
+<div class="general">
+=== Inoculation of BL21 (pASK75 (SA1)) culture in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline  ===
-<span style="color:#2e74b5;">'''SDS-PAGE of </span><span style="color:#4471c4;">eGFP-fractions after IEC'''</span>
+'''Investigator: CR'''
-'''Investigator: '''NA, CG
+'''Aim of the experiment:''' Production of streptavidin
-'''Procedure:''' mixing of 80 µl sample with 20 µl SDS buffer and heating at 95°C for 10 min. 1 h staining, 1 night unstaining
+'''Procedure:'''
-Concentrations were measured via nanodrop (A280, extinction coefficient: 55000 M<sup>−1</sup>cm<sup>−1</sup>)
+* Ampicillin (2 mL) was added to the Medium (1:1000)
-'''Result: '''concentrations in mg/ml* 1. Lane: 8 µl Marker
-* 2. Lane: 10 µl of fraction 10; c= 0
-* 3. Lane: 10 µl of fraction 11; c= 0,15
-* 4. Lane: 10 µl of fraction 12; c= 0,77
-* 5. Lane: 10 µl of fraction 13; c= 0,29
-* 6. Lane: 10 µl of fraction 14; c= 0,4
-* 7. Lane: 10 µl of fraction 15; c= 0,144
-* 8. Lane: 10 µl of fraction 16; c= 0,085
-<div style="margin-left:0.5in;margin-right:0in;">eGFP is clearly detectable at about 27 kDa. The most pure fractions 12 and 13 were pooled for later biotinylation.</div>
-[[Image:|top]]
-<div style="text-align:center;"><span style="color:#6fac47;">'''Friday, May 27</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
-<span style="color:#2e74b5;">'''Inoculation of pre-cultures with B0034, K577894, KK577895, F13+F14'''</span>
-'''Investigator: '''JH
-'''Procedure: '''* Add 50 µL ampicillin(B0034)/chloramphenicol(K5777894, K577895, F13+F14) to 50 mL LB-medium
-* Picking colonies from plate (XL1 blue; all rescue)
-* Incubate over night at 37°C
-*
-<span style="color:#2e74b5;">'''Inoculation of BL21 (pASK75 (SA1)) </span><span style="color:#2e74b5;">culture</span><span style="color:#2e74b5;"> in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline'''</span>
-'''Investigator: '''NA
-'''Aim of the experiment:''' Production of streptavidin
-'''Procedure:''' * Ampicillin (2 mL) was added to the Medium (1:1000)
 * The pre-culture (50 mL) was poured into the Medium
-* Culture was incubated at 37°C and 140 rpm until OD<sub>550</sub> reached 0.5
+* Culture was incubated at 37°C and 140 rpm until OD550 reached 0.5
 * To induce streptavidin expression anhydro-tetrazycline (200 µL) was added to the culture (1:10000)
 * The culture was incubated at 37°C and 140 rpm for 4 hours
+'''Results:'''
+* Streptavidin expression by BL21
+</div>
+<div class="general">
+=== Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium   ===
-'''Result:'''* Streptavidin expression by BL21
+'''Investigator: CR, JB, JH '''
+'''Aim of the experiment:''' Recombinant expression and purification of Streptavidin
+'''Procedure:'''
+* After expression, cultures were transferred into centrifuge tubes and spun down in the centrifuge (4°C, 5000 rpm, 20 mins, F 4X1L rotor)
+* The supernatant was cast away and the pellet was transferred into a beaker of sufficient size and resuspended in fridge-cooled Tris Buffer B (50 mM Tris/HCl (pH = 8.0), 1 mM EDTA)
+* The solution was homogenized in the PANDA (ask supervisor)
+* The resulting lysate was transferred into centrifuge tubes and spun down (4°C, 18,000 rpm, 10 mins, XX34-rotor). The supernatant was cast away and the pellet was resuspended in 6M Gua-HCl (pH = 1.5) at 4°C overnight.
+</div>
+<div class="general">
+=== Dialysis of eGFP ===
+'''Investigator: NA, JH, CR '''
+'''Aim of the experiment:''' Purification of eGFP
+'''Procedure:'''
+* eGFP was thawed on ice
+* eGFP was then poured in a dialysis hose (cut-off 14 kDa)
+* The hose was then placed in ice cold Tris/HCl 20 mM pH 8.0
+* The dialysis took place at 4°C over nightXX34-rotor). The supernatant was cast away and the pellet was
+</div>
-<span style="color:#2e74b5;">'''Sequencing of P30 (again)'''</span>
+<div class="general">
+=== MiniPrep of quickchanged pNGAL146-A2  ===
-'''Investigator: '''NA
+'''Investigator: NA '''
-'''Aim of the experiment:'''
+'''Aim of the experiment:''' Extraction of pNGAL146-A2 plasmid from XL1 blue
-Purification and sequencing of P30
+'''Procedure:'''
-'''Procedure:'''* P30 was heated up to 100 °C and centrifugated afterwards to remove proteins from Promotor (if there are some), the supernatant is sequenced
+* MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-* Sequencing batches were prepared after manufacturer's protocol
+</div>
+<div class="general">
+=== Sequencing of P14, P15 & P19  ===
+'''Investigator: CR, NA '''
-(15 μL plasmid DNA (50-100 μM) and 2 μL sequencing primer)
+'''Aim of the experiment:''' Sequencing of P14, P15 & P19
-The different plasmids we prepared received the following barcodes:
+'''Procedure:'''
-P30 (VF2) : FR11326638
+* Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)
+* The different plasmids we prepared received the following barcodes:
+* P14        : FR11326653
+* P15        : FR11326655
+* P19 (K4): FR11326654
+* P16 (K1): FR11326652
+* P17 (K2): FR11326651
+* P18 (K3): FR11326650
+</div>
+<div class="general">
+=== Digestion of P16, P17, P18 & P19 with AgeI & HindIII + analytical gel  ===
+'''Investigator: NA, JH, CR '''
+'''Aim of the experiment:''' Verification of success of quickchange
-<span style="color:#2e74b5;">'''Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium '''</span>
+'''Procedure:'''
-'''Investigator: '''JH, NA
+* analytic digestion with: 0,25 µl HindIII (HF), 0,25 µl AgeI (HF), 1 µl SmartCut Buffer, 500 ng plasmid-DNA, fill up with ddH2O (Vtotal= 10µL)
+* 10 µl on 2% agarose gel for electrophoresis
-'''Aim of the experiment:''' Recombinant expression and purification of Streptavidin
+'''Results:''' No signal at 600 bp --> quickchange seems to be successful (waiting for sequencing)
+</div>
-'''Procedure:''' * After expression, cultures were transferred into centrifuge tubes and spun down in the centrifuge (4°C, 5000 rpm, 20 mins, F 4X1L rotor)
+=='''Thursday, May 19th'''==
-* The supernatant was cast away and the pellet was stored at –20 °C
+<div class="general">
+===Re-Sequencing of P19===
+'''Investigator: CR'''
+'''Aim of the experiment:''' Re-Sequencing of P19
+'''Procedure:'''Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)
+The different plasmids we prepared received the following barcodes:
+*P19 (K4): FR11326649
+</div>
+<div class="general">
+===Cloning of A3C5 and Avi-Tag into pSB1C3===
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
+'''Investigator: CG'''
-'''Investigator:''' CG
+'''Aim of the experiment:''' Re-Trafo of pSB1C3 RFP for later on: digestion, dephosphorylation and cloning
-'''Aim of the experiment:''' MiniPrep of cloned pSB1C3-A3C5 and –AviTag Plasmids
+'''Procedure:''' transformation according to protocol of P4 E. Coli XL1
-'''Procedure:''' * MiniPrep of inoculated precultures was performed according to manufacturer’s protocol (QIAprep MiniPrep, Qiagen)
+'''Result:''' plates (LB Cam) in incubator for further processing (37 °C)
-* Concentrations were measured (in ng/µL):
+</div>
+<div class="general">
+===Streptavidin refolding===
+'''Investigator: JB'''
+'''Aim of the experiment:''' Refolding of denaturated Streptavidin
+'''Procedure:''' After the pellet had almost completely dissolved in 6M GdmCl, the solution was spun down (4°C, 20 mins, 18,000 rpm). The supernatant was transferred carefully into a falcon tube and the pellet was cast away. Via a hydraulic pump (flow rate: 2x10 ml/min) the lysate was transferred Into 5L PBS 1x. Afterwards the pump was cleaned with technical isopropanol and ELGA water. The solution was stirred overnight at 4°C for refolding.
+</div>
-Colony 1 of pSB1C3- AviTag P32: 120
+<div class="general">
+===Biotinylation of BSA===
-Colony 2 of pSB1C3- AviTag P33: 110
+'''Investigator: JB'''
-Colony 1 of pSB1C3-A3C5 P34: 126
+'''Aim of the experiment:''' Biotinylation of BSA
-Colony 2 of pSB1C3-A3C5 P35: 188
+'''Procedure:''' A 100 µM (=6.8 mg/ml) solution of BSA (Albumin fraction V, pH=7, in the fridge in the central lab) was created (V=10 ml). 220 µl of a 100 mM Biotin stock were added. The mixture was stored overnight Iin the fridge (4°C).
+'''Result:''' Hopefully biotinylated BSA mixture in the fridge (4°C).
+</div>
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
-'''Investigator: '''NA, JH, CG
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
-'''Aim of the experiment:''' Initial vector digestion was performed with NgoMiV and AgeI, producing complementary overhangs. Religated pSB1C3 (due to incomplete vector dephosphorylation) loses the AgeI recognition sequence. Therefore plasmids without insert should run in the supercoiled position at about 1.5 kb, plasmids with inserts should run in the linearised position at 3 kb. Single enzyme digestion was chosen, because the insert is too small to be solved on a gel.
+=Week 2 (May 23rd - May 29th=
+<div class="week" id="WWeek_2">
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+=Week 3 (May 30th - June 5th=
+<div class="week" id="WWeek_3">
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
-'''Procedure:''' * analytic digestion: 3 µl of plasmid DNA P32 to P35 (about 300 ng) were digested with 0.3 µl AgeI-HF in 1 µl CutSmart Buffer and 5.7 µl H2O. The reaction was incubated for 2 h at 37°C.
+=Week 4 (June 6th - June 12th)=
-* 5 µl digestion mix were loaded on a 1% agarose gel for electrophoresis, 1 kb ladder
+<div class="week" id="WWeek_4">
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+=Week 5 (June 13th - June 19th)=
+<div class="week" id="WWeek_5">
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+=Week 6 (June 20th - June 26th)=
+<div class="week" id="WWeek_6">
+=='''Thursday, June 23rd'''==
+<div class="receptor">
-'''Result:'''
+===Miniprep of E. coli Xl1-Blue transformed with ligation product P80/81 (mRuby3 K1/2), P82/83 (EspP K1/2), P84/85 (StrepTag K1/2) and Trafo of K157001===
-P32 to P35 might carry the insert because they all appear in the linearized position at 3 kb. Therefore plasmid sequencing should confirm the result.
+'''Investigator: Jan, Julian'''
+'''Aim of the experiment:''' Miniprep of E. coli Xl1-Blue transformed with ligation product F50(K1,2), F51(K1,2), F52(K1,2) and Trafo of K157001
+'''Procedure:'''
-Lane 1: 1 kb ladder
+* Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
+* Concentrations:
+<table class="wikitable" cellspacing="0" border="1">
+<tr>
+<td><b>Plasmid</b>
+</td><td><b>c [ng/µl]</b>
+</td></tr>
+<tr>
+<td>P80
+</td><td>432,7
+</td></tr>
+<tr>
+<td>P81
+</td><td>294,8
+</td></tr>
+<tr>
+<td>P82
+</td><td>450,5
+</td></tr>
+<tr>
+<td>P83
+</td><td>479,0
+</td></tr>
+<tr>
+<td>P84
+</td><td>108,0
+</td></tr>
+<tr>
+<td>P85
+</td><td>356,0
+</td></tr><tr>
+<td>P86
+</td><td>47,2
+</td></tr></table>
+</div>
-Lane 3: P32
+<div class="Receptor">
-Lane 4: P33
+=='''Friday, June 24th'''==
-Lane 5: P34
+<div class="receptor">
-Lane 6: P35
+=== Analytical digestion and gelelectrophoresis of P83 (EspP K2) and P83 (StrepTag K2) ===
+'''Investigator: Julian, Niklas, Luisa'''
-[[Image:|top]]
+'''Aim of the experiment:''' Analytical digestion and gelelectrophoresis of P82/83 (EspP K1/2) and P84/85 (StrepTag K1/2).
+'''Procedure:'''
+* Batch for analytical digestion for P82-P85 with EcoRI-HF
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|0.5/1.0&nbsp;µl
+|Plasmid DNA (-/P84)
+|-
+|1&nbsp;µl
+|CutSmart buffer (10x)
+|-
+|0.5&nbsp;µl
+|EcoRI-HF(10&nbsp;U/µl)
+|-
+|8/7.5&nbsp;µl
+|ddH2O (-/P84)
+|-
+|=10&nbsp;µl
+|'''TOTAL'''
+|}
+[[File:Muc16_P82-85_EcoRI.png|500px]]
+</div>
+<div class="receptor">
-<div style="text-align:center;"><span style="color:#6fac47;">'''Monday, May 30</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
+=== Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)===
+'''Investigator: Julian'''
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
+'''Aim of the experiment:''' Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)
-'''Investigator: '''CR
-'''Aim of the experiment:'''
-Sequencing of P32 and P35
 '''Procedure:'''
-Sequencing batches were prepared according to manufacturers protocol (Mix2Seq Kit, Eurofins Genomics): 15 μL plasmid DNA (50-100 μM) and 2 μL sequencing primer (10 µM)
+Sequencing batch were prepared after manufacturer's protocol. (15&nbsp;µl of plasmid DNA (50 - 100&nbsp;ng) and 2&nbsp;µl sequencing primer). Sequencing primer VF2 was used
+The different vectors we sequenced received the following barcodes:
-The plasmids received the following barcodes:
+* mRuby3 in pSB1C3 (P80):   FR11326590
-P32 (VF2) : FR11326637
+* EspP in pSB1C3 (P83):  FR11326588
-P35 (VF2) : FR11326636
+* Streptag in pSB1C3 (P85):  FR11326587
+</div>
+<div class="receptor">
+===  Transformation of ''E.&nbsp;coli'' XL1 blue with F64 (quickchanged P3(pSAm1)) ===
-<span style="color:#2e74b5;">'''Religation of F13 and transformation in XL1 blue'''</span>
+'''Investigator: Niklas'''
-'''Investigator: '''CR
+'''Aim of the experiment:''' Transformation of ''E.&nbsp;coli'' XL1 blue.
-'''Aim of the experiment:'''
+'''Procedure:'''
-Re-ligate digested and dephosphorylated pSB1C3 and transformation in XL1blue
+* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
-'''Procedure:'''* Plasmid was ligated with the T4 DNA Ligase following the manufacturer’s protocol (NEB)
+* 10&nbsp;µl of DNA was added to 100&nbsp;µl of competent cells and gently mixed.
-* 52 ng Vector DNA were used
-* The Ligation mix was incubated for 30 min at RT
-* 7 μL Ligation mixed were used for the transformation of 50 μL XL1 blue.
-* Transformation was performed following the SOP
+* 30&nbsp;min incubation on ice
+* 5&nbsp;min. heat shock at 37&nbsp;°C
-'''Result:'''* 5 clones on plate
+* Adding of 750&nbsp;µl LB-medium to each tube.
+* The cell suspension was plated on ampicillin plates (inclusive rescue plate) and incubated over night at 37&nbsp;°C in the cell-culture shaker.
+--> Quickchange did not work, do again, than new transformation!
+</div>
+<div class="receptor">
+===  Gelextraction of F67(BirA), F68(mRuby), F69(EGFR-TMD), F70(pSB1C3) and F71(pSB1C3) ===
+'''Investigator: Niklas'''
+'''Aim of the experiment:''' Gelextraction of F67(BirA(Digest. F59 [EcoRI; SpeI])), F68(mRuby(Digest. F60 [NgoMIV; SpeI])), F69(EGFR-TMD(Digest. F60 [NgoMIV; SpeI]), F70(pSB1C3(digest. P74 [NgoMIV; SpeI]) and F71(pSB1C3(digest. P74 [EcoRI; SpeI])
-<span style="color:#2e74b5;">'''Inoculation of pre-cultures with B0034, K577894, KK577895, F13+F14'''</span>
+'''Procedure:'''
-'''Investigator: '''CR
+Gelextraction was performed by manufacturers protocol (Qiagen).
+</div>
-'''Procedure: '''* Add 50 µL ampicillin(B0034)/chloramphenicol(K5777894, K577895, F13+F14) to 50 mL LB-medium
+=='''Saturday, June 25th'''==
-* Picking colonies from plate (XL1 blue; all rescue)
+<div class="Receptor">
-* Incubate over night at 37°C
+===Miniprep of E. coli Xl1-Blue transformed with P60 (mRuby/EGFR),F58 (Ligation pASK75 + Streptactin), F65 (CMV + CD4), F66 (CMV + EGFR), P70 (Short Linker)===
+'''Investigator: Niklas'''
+'''Procedure:'''
+* Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
+* Concentrations:
+<table cellspacing="0" border="1">
+<tr>
+<td><b>Plasmid</b>
+</td><td><b>c [ng/µl]</b>
+</td></tr>
+<tr>
+<td>P87
+</td><td>81
+</td></tr>
+<tr>
+<td>P88
+</td><td>34,5
+</td></tr>
+<tr>
+<td>P89
+</td><td>86,3
+</td></tr>
+<tr>
+<td>P90
+</td><td>108,5
+</td></tr>
+<tr>
+<td>P91
+</td><td>417,4
+</td></tr></table>
+</div>
+<div class="Receptor">
+=== Analytical digestion and gelelectrophoresis of F64 (quickchanged P3) for verification of succesful quickchange ===
+'''Investigator: Niklas'''
-<div style="text-align:center;"><span style="color:#6fac47;">'''Tuesday, May 31</span><span style="color:#6fac47;"><sup>st'''</sup></span></div>
+'''Procedure:'''
+* Analytical digestion with NdeI and gelelectrophoresis. If quickchange worked there should be a band at about 3200 bp (only one restriction site left)
+* Incubation over night at room temperature.
+* Analytical gelelectrophoresis was performed at 90&nbsp;V for 60&nbsp;min.
-<span style="color:#2e74b5;">'''Dialysis of eGFP'''</span>
-'''Investigator: '''NA
-'''Aim of the experiment:''' Changing the buffer of eGFP for biotinylation (from TRIS to PBS)
-'''Procedure:''' * eGFP was poured in a dialysis hose (cut-off 14 kDa)
-* The hose was then placed in ice cold PBS pH 7.4
-* The dialysis took place at 4°C overnight
-<span style="color:#2e74b5;">'''Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium '''</span>
-'''Investigator: '''JB, NA
-'''Aim of the experiment:''' Recombinant expression and purification of Streptavidin
-'''Procedure:''' * The pellet was transferred into a beaker of sufficient size and resuspended in fridge-cooled Tris Buffer B (50 mM Tris/HCl (pH = 8.0), 1 mM EDTA)
-* The solution was homogenized in the PANDA (see SOP)
-* The resulting lysate was transferred into centrifuge tubes and spun down (4°C, 18,000 rpm, 30 mins, SS34-rotor). The supernatant was cast away and the pellet was resuspended in 6M Gua-HCl (pH = 1.5) at 4°C overnight.
-<span style="color:#2e74b5;">'''Transformation of P30 '''</span>
-'''Investigator: '''NA
-'''Aim of the experiment:''' expression of tet-repressed GFP in E. Coli BL21
-'''Procedure:''' transformation in competent E. Coli BL21 according to protocol
-'''Result: '''plates (LB Cam) in incubator for further processing (37 °C)
-<span style="color:#2e74b5;">'''Cloning of A3C5 and Avi-Tag into pSB1C3'''</span>
-'''Investigator: '''CR, JH
-'''Aim of the experiment:'''
-Preculture of pSB1C3 (F13+F14) was red
-See if RFP-generator is still inside the pSB1C3
-See if ligation of EspP was successful
-'''Procedure:''' * MiniPrep of P39 was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-* pSB1C3 was digested with AgeI and NgOMIV (1.5 µL P39 + 1.5 µL AgeI + 1.5 µL NgoMIV + 3 µL CutSmart + 22.5 µL ddH<sub>2</sub>O; incubation for 2h, 37°C)
-* FastAP was added and the reaction was incubated for another 10min at 37°C
-* FastAP was inactivated by heating the mix to 75°C for 10min.
-* The digestion was purified by gelelctrophoresis (1%, 75V, 1h)
 '''Results:'''
+[[File:Muc16_Quickchange_NA.JPG]]
-[[Image:|top]]
+Just a band showing a few bp (Primer), there is no plasmid band -> Quickchange did not work
+</div>
+<div class="Receptor">
-* Unknown signals.
+=== Inoculation of colonies from Ligation of F69 + F70 (EGFR-TMD in pSB1C3)and F44 + F30 (mRuby in pSB1C3) ===
-* Repeat transformation, digestion, dephosphorylation and ligation of pSB1C3
+'''Investigator: Niklas'''
+'''Procedure:'''
+* 6x 4 ml LB+Cam media
+* Each culture was inoculated with one colony
+* Incubation at 37°C overnight
+</div>
-<span style="color:#2e74b5;">'''Re-transformation of P4 (pSB1C3 (RFP-generator)) in XL1 blue'''</span>
+=='''Sunday, June 26th'''==
-'''Investigator: '''JH, CR
+<div class="Receptor">
-'''Aim of the experiment:''' Transformation
+===Miniprep of E. coli Xl1-Blue transformed with ligation product F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3) ===
-'''Procedure:''' * 2 μL P4 were used for transformation of XL1 blue
+'''Investigator: Luisa'''
-* Transformation was performed according to SOP (incl. rescue plate)
+'''Aim of the experiment:''' Extracting F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3) from E.coli XL-1-blue
+'''Procedure:'''
+* Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
+* Concentrations:
+<table cellspacing="0" border="1">
+<tr>
+<td><b>Plasmid</b>
+</td><td><b>c [ng/µl]</b>
+</td></tr>
+<tr>
+<td>P92
+</td><td>162,9
+</td></tr>
+<tr>
+<td>P93
+</td><td>447,9
+</td></tr></table>
+</div>
+<div class="Receptor">
+=== Repetition of Quick-Change PCR of P3 (pASK + SAm1) ===
-<span style="color:#2e74b5;">'''Competent E. coli XL1 cells'''</span>
+'''Investigator: Luisa'''
-'''Investigator: '''JB
+'''Procedure:'''
-'''Aim of the experiment:''' Generation of competent E.coli XL1 cells
+* The QC-PCR was performed according the SOP.
-'''Procedure: '''* 5 ml LB w.o. antibiotics (sterile)
+* Reaction Mix:
-* Inoculation of one colony of E.coli XL1 cells
+<table cellspacing="0" border="1">
-* Incubate overnight at 37°C
+<tr>
+<td><b>volume</b>
+</td><td><b>reagent</b>
+</td></tr>
+<tr>
+<td>1,25 µl
+</td><td>Primer O21
+</td></tr>
+<tr>
+<td>1,25 µl
+</td><td>Primer O22
+<tr>
+<td>1 µl
+</td><td> dNTP-mix
+</td></tr>
+<tr>
+<td> 5 µl
+</td><td>Pfu-Ultra-II reaction buffer
+<tr>
+<td>1 µl
+</td><td> template DNA (1:10 dilution of p3)
+</td></tr>
+<tr>
+<td> 0,5 µl
+</td><td>Pfu-Ultra-II Polymerase
+<tr>
+<td>40,5 µl
+</td><td> ddH2O
+</td></tr></table>
+* digestion of PCR-Product with DpnI for 1h at 37°C
+* now labeled P94
+</div>
+===  Transformation of ''E.&nbsp;coli'' XL1 blue with P94 (quickchanged P3(pSAm1)) ===
+'''Investigator: Luisa'''
+'''Aim of the experiment:''' Transformation of ''E.&nbsp;coli'' XL1 blue.
+'''Procedure:'''
+* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were put out from the stock in -80&nbsp;°C freezer and were gently thawed on ice.
+* 10&nbsp;µl of DNA was added to 100&nbsp;µl of competent cells and gently mixed.
-<div style="text-align:center;"><span style="color:#6fac47;">'''Wednesday, June 1</span><span style="color:#6fac47;"><sup>st'''</sup></span></div>
+* 30&nbsp;min incubation on ice
+* 5&nbsp;min. heat shock at 37&nbsp;°C
-<span style="color:#2e74b5;">'''eGFP Biotinylation'''</span>
+* Adding of 950&nbsp;µl LB-medium to each tube.
-'''Investigator: '''CG
+* The cell suspension was plated on ampicillin plates (inclusive rescue plate) for pASK (F72) and on chloramphenicol plates for P92 and P93 and incubated over night at 37&nbsp;°C in the incubator.
-'''Aim of the experiment:''' Biotinylation of dialysed eGFP past IEC
+<div class="Receptor">
-'''Procedure:''' * The average concentration of the pooled factions 12 and 13 should be about 0.5 mg/ml (=19 µM)
+=== Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75)  ===
-* 10 ml of a 100 mM Biotin stock-solution was prepared (340 mg)
-* 3.5 ml of eGFP were biotinylated with 12 µl 100 µM Biotin-solution (20x molar excess)
-* Incubation at 4 °C overnight
+'''Investigator: Luisa'''
+'''Aim of the experiment:''' Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75).
+'''Procedure:'''
-<span style="color:#2e74b5;">'''pSB1C3 vector backbone preperation'''</span>
+* Batch for analytical digestion for P92 and P93 with EcoRI-HF and PstI-HF
+{|cellspacing="0" border="1"
-'''Investigator: '''CG
+|'''volume'''
+|'''reagent'''
-'''Aim of the experiment:''' Inoculation of two colonies pSB1C3-RFP Generator
+|-
+|1&nbsp;µl
-'''Procedure: '''* 2x 5 ml LB+Cam medium (1:1000)
+|Plasmid DNA
-* Picking colonies from plate (XL1 blue, rescue)
+|-
-* Incubate overnight at 37°C
+|1&nbsp;µl
+|CutSmart buffer (10x)
+|-
+|0.5&nbsp;µl
+|EcoRI-HF(10&nbsp;U/µl) and PstI-HF (10&nbsp;U/µl) for P92/93, NdeI (10&nbsp;U/µl) for P94
+|-
-<span style="color:#2e74b5;">'''Generation of GFP expressing E. coli cells'''</span>
+|7.5/7&nbsp;µl
+|ddH2O
-'''Investigator: '''CG
+|-
+|=10&nbsp;µl
-'''Aim of the experiment:''' Inoculation of BL21 transformed cell with P30
+|'''TOTAL'''
+|}
-'''Procedure: '''* 50 ml LB+Cam medium (1:1000)
-* Picking colonies from plate (BL21, rescue)
-* Incubate over night at 37°C
-<span style="color:#2e74b5;">'''Competent E. coli XL1 cells'''</span>
-'''Investigator: '''JB
-'''Aim of the experiment:''' Generation of competent E. coli XL1 cells
-'''Procedure: '''* Inoculated 500 µl preculture in 50 ml LB media w.o. antibiotics
-* Incubated and shaken at 37°C until the OD550 reached 0.44
-* Transferred into a falcon tube and incubated on ice for 10 mins
-* Spun down at 3000 g for 10 mins at 4°C, supernatant cast away
-* Pellet resuspended in 40 ml fridge-cooled MgCl2 (100 mM), once again spun down at 4°C for 10 mins at 3000 g
-* Supernatant cast away, pellet resuspended in 20 ml cold CaCl2 (50 mM)
-* Incubation on ice for 30 mins
-* Centrifugation repeated, supernatant cast away
-* Pellet resuspended in 2 ml of CaCl2 (50 mM) and glycerol (15%)-solution, aliquoted (50 µl each), frozen in liquid nitrogen and stored at -80°C for further use
-<span style="color:#2e74b5;">'''Cloning of Pcat-RBS-construct in pSB1A2'''</span>
-'''Investigator: '''CG, JH, CR
-'''Aim of the experiment:''' Pcat (I14033) was cut out of P27 and ligated into P38 for generating the Pcat-RBS (B0034) construct.
-'''Procedure:''' * P27 was digested with EcoRI and SpeI (4 µL P27 + 1.5 µL EcoRI + 1.5 µL SpeI + 3 µL CutSmart + 20 µL ddH<sub>2</sub>O; incubation for 2h, 37°C)
-* P38 was digested with EcoRI and XbaI (10 µL P27 + 1.5 µL EcoRI + 1.5 µL XbaI + 3 µL CutSmart + 14 µL ddH<sub>2</sub>O; incubation for 2h, 37°C)
-* Preperative gel electrophoresis was performed
-* For P27 the signal at about 50 bp was cut out and gel extracted according to manufacturer's protocol (Gelextractionkit, Qiagen): 5.9 ng/µl
-* For P30 the signal at about 2.1 kb was cut out and gel extracted according to manufacturer's protocol (Gelextractionkit, Qiagen): 8.9 ng/µl
-* Ligation according to SOP
-<span style="color:#2e74b5;">'''Purification of streptavidin'''</span>
-'''Investigator: '''JB
-'''Aim of the experiment: '''Refolding denaturized streptavidin
-'''Procedure: '''
-'''- '''the GdmCl-solved streptavidin was spun down using an SS-34 rotor (~60 min, 18,000 rpm, 4°C)
-- The supernatant containing the solved and denaturized protein was transferred into a falcon tube, the pellet cast away
-- The protein solution was slowly dripped into 4 l of cooled 1x PBS (in the 4°C room) using a hydraulic pump and stirred overnight
-<div style="color:#6fac47;"></div>
-<div style="text-align:center;"><span style="color:#6fac47;">'''Thursday, June 2</span><span style="color:#6fac47;"><sup>nd'''</sup></span></div>
-<span style="color:#2e74b5;">'''Preparation of pSB1C3 for further use'''</span>
-'''Investigator: '''CR
-'''Aim of the experiment:''' Digestion of pSB1C3 to remove RFP-generator
-'''Procedure:''' * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
-* P40 was digested with AgeI and NgOMIV (2.5 µL P40 + 1.5 µL AgeI + 1.5 µL NgoMIV + 3 µL CutSmart + 21.5 µL ddH<sub>2</sub>O)
-* Incubation over night at 37°C
-<span style="color:#2e74b5;">'''Generation of GFP expressing E. coli cells'''</span>
-'''Investigator: '''CG, JH
-'''Aim of the experiment:''' Various inductor concentrations
-'''Procedure: '''* 12x 5 ml LB+Cam media were inoculated with 50 µl preculture
-* when cultures reached OD550 of 0.5, they were induced with tetrazycline-anhydride of various concentrations (0 ng/µl, 20 ng/µl, 40 ng/µl etc., 200 ng/µl)
-* t=0, =30, =90, =150, =210, =270 a 100 µl aliquot of each culture was stored at 4 °C in the refrigerator
-<span style="color:#2e74b5;">'''Dialysis of biotinylated eGFP'''</span>
-'''Investigator:''' JH, JB
-'''Aim of the experiment:''' Purification of biotinylated eGFP
-'''Procedure:''' * eGFP was poured in a dialysis hose (cut-off 14 kDa)
-* The hose was then placed in 2 L ice cold Tris/HCl 20 mM pH 8.0
-* The dialysis took place at 4°C over night
-<span style="color:#2e74b5;">'''Ligation of F17.1 and F18 and transformation in XL1 blue'''</span>
-'''Investigator: '''JH
-'''Aim of the experiment:'''
-Ligation and transformation in XL1blue
-'''Procedure:'''* Plasmid was ligated with the T4 DNA Ligase following the manufacturer’s protocol (NEB)
-* 9 µL F18 (B0034 in pSB1A2; RBS) and 1 µL of F17.1 (I14033; P(cat)) were used [AmpR]
-* The Ligation mix was incubated for at least 2h at RT
-* 7 μL Ligation mixed were used for the transformation of 50 μL XL1 blue
-* Transformation was performed following the SOP [AmpR], incl. rescue plate
-<div style="text-align:center;"><span style="color:#6fac47;">'''Friday, June 3</span><span style="color:#6fac47;"><sup>rd'''</sup></span></div>
-<span style="color:#2e74b5;">'''Cloning of F8, F9 and F14 into F20'''</span>
-'''Investigator: '''CR
-'''Aim of the experiment:''' Cloning of A3C5, Avi-Tag and into pSB1C3
-'''Procedure:''' * FastAP was added and the digestion of F20 and was incubated for another 15 min at 37°C
-* The digestion was purified by gelelctrophoresis (1%, 75V, 1h)
-* Plasmid backbone (signal 1) was cut from the gel
-* Gelextraction was performed after manufacturer's protocol (Gelextractionkit, Qiagen)
-<div style="margin-left:0.5in;margin-right:0in;"></div>
 '''Results:'''
+* 1. band (P92): no mRuby at 700bp visible, only empty vector
+* 2. band (P93): empty vector and EGFR --> perfect
+* 3. band (P94): no signal at all --> repetition of QC-PCR
-[[Image:|top]]* Signal 1: F20
-* [F20]= 13,8 ng/μL
+</div>
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+=Week 7 (June 27th - July 3rd)=
+<div class="week" id="WWeek_7">
+=='''Monday, June 27th'''==
+=== Sequencing of P67 (EGFR-Signalpeptid) ===
+'''Investigator: Niklas'''
-<span style="color:#2e74b5;">'''Generation of GFP expressing E. coli cells'''</span>
+'''Procedure:'''
-'''Investigator: '''CG, NA
+Sequencing batch was prepared after manufacturer's protocol. (15&nbsp;µl of plasmid DNA (100&nbsp;ng) and 2&nbsp;µl sequencing primer (VF2))
-'''Aim of the experiment:''' SDS PAGE for verification of successful GFP expression
+FR11326586
+</div>
-'''Procedure: '''* SDS electophoresis according to SOP
+<div>
-* Lane 1: #12, 90 min
-* Lane 2: #12, 150 min
-* Lane 3: #12, 210 min
-* Lane 4: #12, 270 min
-* Lane 5: 8 µl Ladder
+<div>
+=== Repetition of Quick-Change PCR of P3 (pASK + SAm1) ===
+'''Investigator: Luisa'''
+'''Procedure:'''
+* The QC-PCR was performed according the SOP.
+* Reaction Mix:
+<table cellspacing="0" border="1">
+<tr>
+<td><b>volume</b>
+</td><td><b>reagent</b>
+</td></tr>
+<tr>
+<td>1,25 µl
+</td><td>Primer O21
+</td></tr>
+<tr>
+<td>1,25 µl
+</td><td>Primer O22
+<tr>
+<td>1 µl
+</td><td> dNTP-mix
+</td></tr>
+<tr>
+<td> 5 µl
+</td><td>Pfu-Ultra-II reaction buffer
+<tr>
+<td>1 µl
+</td><td> template DNA (1:10 dilution of p3)
+</td></tr>
+<tr>
+<td> 0,5 µl
+</td><td>Pfu-Ultra-II Polymerase
+<tr>
+<td>40,5 µl
+</td><td> ddH2O
+</td></tr></table>
+* Digestion of PCR-Product with DpnI for 1h at 37°C.
+* Transformation of 10µl into component E.coli XL-1-blue, according to SOP (1h incubation at 37°C necessary despite AmpR).
+</div>
+=== PCR of Genesynthesis 3 and 4 ===
+'''Investigator: Luisa'''
+'''Aim of Experiment: Amplification of Genesynthesis 3 (contains BAP and IGKappa) and 4 (contains A3C5-tag and BM40)'''
+'''Procedure:'''
+* The PCR was performed according the SOP.
-Result:
+* Reaction Mix:
+<table cellspacing="0" border="1">
+<tr>
+<td><b>volume</b>
+</td><td><b>reagent</b>
+</td></tr>
+<tr>
+<td> 2,5 µl
+</td><td>Primer VF2
+</td></tr>
+<tr>
+<td> 2,5 µl
+</td><td>Primer VR2
+<tr>
+<td> 1 µl
+</td><td> dNTP-mix
+</td></tr>
+<tr>
+<td> 10 µl
+</td><td> Q5 Polymerase reaction buffer
+<tr>
+<td>1 µl
+</td><td> template DNA (1:10 dilution of p3)
+</td></tr>
+<tr>
+<td> 0,5 µl
+</td><td> Q5-Polymerase
+<tr>
+<td> 18 µl
+</td><td> ddH2O
+</td></tr></table>
-[[Image:|top]]
+*Setup: iGEM_standard (Promega-cycler)
+<table cellspacing="0" border="1">
+<tr>
+<td><b>temperature</b>
+</td><td><b>time</b>
+</td></tr>
+<tr>
+<td> 98°C
+</td><td> 2min
+</td></tr>
+<tr>
+<td> 98°C
+</td><td> 10sec
+<tr>
+<td> 66°C
+</td><td> 30sec
+</td></tr>
+<tr>
+<td> 72°C
+</td><td> 30sec
+<tr>
+<td> 72°C
+</td><td> 2min
+</td></tr>
+<tr>
+<td> 4°C
+</td><td> hold
+</td></tr></table>
+* the batches were then purified using the Quiagen PCR-Purification Kit.
+</div>
+=== Analytical digestion and gelelectrophoresis of P88 , P89 and P90 ===
+'''Investigator: Niklas'''
+'''Aim of experiment: Analytical digestion and gelelectrophoresis of P88 (pASK75 + Streptactin, former F58), P89 (CMV + CD4, former F65) and P90 (CMV + EGFR-signal-peptide, former F66)'''
-<span style="color:#2e74b5;">'''Inoculation of colonies from F19'''</span>
+'''Procedure:'''
+* Batches for analytical digestions:
-'''Investigator: '''CG
+P88: EcoRI
-'''Aim of the experiment:''' three colonies were picked from yesterdays transformation
+P89: EcoRI and PstI
-'''Procedure: '''* 3x 5 ml LB+Amp media
+P90: EcoRI
-* Each culture was inoculated with one colony
+{|cellspacing="0" border="1"
-* Incubation at 37°C overnight
+|'''volume'''
+|'''reagent'''
+|-
+|5,8/2,3/1,8 &nbsp;µl
+|Plasmid DNA (P88/P89/P90)
+|-
+|1&nbsp;µl
+|CutSmart buffer (10x)
+|-
+|0.5&nbsp;µl
+|EcoRI-HF(10&nbsp;U/µl)/ PstI
+|-
+|required amount for total volume of 10&nbsp;µl
+|ddH2O
+|}
+[[File:Muc16_P88-90_NA.JPG]]
+===  Ligation of F67 and F71, Transformation of ''E.&nbsp;coli'' XL1 blue afterwards ===
+'''Investigator: Niklas'''
+'''Aim of the experiment:''' Ligation of F67 (BirA) and F71 (empty pSB1C3), Transformation of ''E.&nbsp;coli'' XL1 blue afterwards.
+'''Procedure:'''
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|2,4&nbsp;µl
+|Vektor
+|-
+|7,6&nbsp;µl
+|Insert
+|-
+|2&nbsp;µl
+|10X DNA-Ligase-buffer
+|-
+|1&nbsp;µl
+|T4-Ligase
+|-
+|7&nbsp;µl
+|ddH<sub>2</sub>O
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
+|}
+* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were taken out of stock in -80&nbsp;°C freezer and were gently thawed on ice.
-<span style="color:#2e74b5;">'''Cloning of F21, F22 and F23 into F20'''</span>
+* 7&nbsp;µl of DNA was added to 100&nbsp;µl of competent cells and gently mixed.
-'''Investigator: '''CG
+* 30&nbsp;min incubation on ice
-'''Aim of the experiment:''' Ligation of F21, F22 and F23 into F20 and transformation
+* 5&nbsp;min. heat shock at 37&nbsp;°C
-'''Procedure:''' * Ligation was performed according to the SOP with: 0.7 µl F8 (1:10) / 9.3 µl F20, 0.7 µl F9 (1:10) / 9.3 µl F20, 9.1 µl F14 / 0.9 µl F20
+* Adding of 750&nbsp;µl LB-medium to each tube.
-* Transformation was performed according to the SOP on LB Cam-Agar
+* Incubation for 1 hour at 37&nbsp;°C
+* The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37&nbsp;°C in the cell-culture shaker.
+* next step: analytic digestion of transformation was successful
+</div>
-<span style="color:#2e74b5;">'''Precipitation of Streptavidin'''</span>
+<div>
-'''Investigator: '''NA, JB, LK
+=== Digestion of PCR on genesynthesis 3 and 4, and pSB1C3 ===
-'''Aim of the Experiment: '''Purification of Streptavidin
+'''Investigator: Luisa'''
-'''Procedure:'''* Slowly add (NH<sup>4+</sup>)<sub>2</sub>SO<sub>4</sub><sup>2-</sup> until the concentration reaches 40% (23,1 g/100mL) while stirring
+'''Aim of experiment: Division of Leptin, IGKappa, A3C5, BM40 and BAP using SapI, HindIII, XbaI, AgeI for both batches.'''
-* Let it stir over night to ensure complete precipitation
-* Centrifuge at 8500 rpm at 4°C for 15min (rotor: SLA1500), dismiss the pellet and use the supernatant for further processing
-* Slowly add (NH<sup>4+</sup>)<sub>2</sub>SO<sub>4</sub><sup>2-</sup> until the concentration reaches 70% (19,1 g/100mL) while stirring
-* Let it stir over night (slowly to avoid foaming)
-* Centrifuge at 8500 rpm at 4°C for 15min (rotor: SLA1500), dismiss the supernatant and use the pellet for further processing
-* Resuspend the protein pellet in a minimum volume of TRIS/HCl; pH 8; 20mM (NO SALT) (6 mL per pellet), the pellets where then united and titrated with Buffer until the solution became clear. Total amount of buffer: 40mL
-* Transfer to 50 mL Falcon Tubes and centrifuge at full speed for 15 min. Filter the supernatant through 45nm sterile filter and keep the pellet (rescue).
+'''Procedure:'''
+* Batches for analytical digestions:
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|1 µl each
+|enzyme (SapI, HindIII, XbaI, AgeI)
+|-
+|5&nbsp;µl
+|CutSmart buffer (10x)
+|-
+|41&nbsp;µl
+|DNA (purified PCR-products of GSY3 and 4)
+|-
+|}
+* Additionally 10µg of the vector P74 was digested with XbaI and AgeI in 100µl batch (2µl of each enzyme, 10µl of Cut-Smart buffer). Digestion was performed over night and purified via gelelectrophoresis and gelextraction according to the manufacturer's protocoll. --> Now labeled F80.
+</div>
+=== Analytical digestion and gelelectrophoresis of P80 , P78 and P85 ===
+'''Investigator: Julian'''
-<div style="text-align:center;"><span style="color:#6fac47;">'''Saturday, June 4</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
+'''Aim of experiment: Analytical digestion and gelelectrophoresis of P80 (mRuby3), P78 (NanoLuc) and P85(Strep-Tag)'''
+'''Procedure:'''
+* Batches for analytical digestions:
-<span style="color:#2e74b5;">'''Inoculation of colonies from F21, F22, F23'''</span>
+*P80 and P78: EcoRI and AgeI
-'''Investigator: '''NA
+*P85: EcoRI and NgoMIV
-'''Aim of the experiment:''' three colonies were picked from each transformation
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|10 &nbsp;µl
+|Plasmid DNA
+|-
+|32 &nbsp;µl
+|ddH2O
+|-
+|5&nbsp;µl
+|CutSmart buffer (10x)
+|-
+|1.5&nbsp;µl
+|each enzyme(10&nbsp;U/µl)/ PstI
+|-
+|50&nbsp;µl
+|'''TOTAL'''
+|}
-'''Procedure: '''* 3x 5 ml LB+Cam media for each fragment
+[[File:Muc16_.JPG]]
-* Each culture was inoculated with one colony
-* Incubation at 37°C overnight
+===  Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3 with Strep-Tag) and Transformation into ''E.&nbsp;coli'' XL1 blue ===
+'''Investigator: Luisa'''
+'''Aim of the experiment:''' Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3+Strep-Tag), Transformation of ''E.&nbsp;coli'' XL1 blue afterwards.
+'''Procedure:'''
+{|cellspacing="0" border="1"
+|'''volume'''
+|'''reagent'''
+|-
+|4,3µl(for F75), 8,2µl (for F76)
+|Vector
+|-
+|12,7µl (F75), 8,8 (F76)
+|Insert
+|-
+|2&nbsp;µl
+|10X DNA-Ligase-buffer
+|-
+|1&nbsp;µl
+|T4-Ligase
+|-
+|=20&nbsp;µl
+|'''TOTAL'''
+|}
-<span style="color:#2e74b5;">'''MiniPrep of F19'''</span>
+*Ligation was incubated at RT for 1,5h.
-'''Investigator: '''NA
+* CaCl2 competent ''E.&nbsp;coli'' XL1-Blue cells were taken out of stock in -80&nbsp;°C freezer and were gently thawed on ice.
-'''Aim of the experiment:''' Extraction of Ligation: F17+F18 (starke RBS+P(cat))
+* 7&nbsp;µl of DNA was added to 100&nbsp;µl of competent cells and gently mixed.
-'''Procedure:'''
+* 15&nbsp;min incubation on ice
-MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)
+* 5&nbsp;min. heat shock at 37&nbsp;°C
-Concentrations: K1= 66,2 ng/µl; K2= 60,2 ng/µl; K3= 104,4 ng/µl
+* Adding of 950&nbsp;µl LB-medium to each tube.
+* Incubation for 1 hour at 37&nbsp;°C
-<div style="text-align:center;"><span style="color:#6fac47;">'''Monday, June 6</span><span style="color:#6fac47;"><sup>th'''</sup></span></div>
+* The cell suspension was plated on Cam-plates (inclusive rescue plate) and incubated over night at 37&nbsp;°C in the incubator.
-<span style="color:#2e74b5;">'''Cloning of TetR from p28 into p31 '''</span>
-'''Investigator: '''LK, JH, NA
-'''Aim of the experiment:''' Cloning TetR in front of weak RBS in pSB1C3
-'''Procedure:''' * Digestion of p28 with EcoR1 and Spe1 (20µl DNA, 2,5 µl each enzyme, 5µl CutSmart buffer, 20µl ddH<sub>2</sub>O) and p31 (4µl DNA, 1µl EcoRI / XbaI, 2µl, 12µl DNA) -> Incubation at 37°C for 2h.
-* <div style="margin-left:0.4957in;margin-right:0in;">Electrophoresis: 1% Agar, 15 min , 70 V (oben p31 und p41, unten p28)</div>
-<div style="text-align:center;">[[Image:|top]]</div>* Gelextraction  concentrations: p28 (F25) = 2,3 ng/µl
-<div style="margin-left:2.95in;margin-right:0in;">p31 (F24) = 7,8 ng/µl</div>* Ligation with 6,3 µl vector (F24) and 1,7 µl Insert (F25) + 1µl Buffer + 1µl Ligase  over night, 16°C
-<div style="margin-left:0.25in;margin-right:0in;"></div>
-<span style="color:#2e74b5;">'''SDS-PAGE of </span><span style="color:#4471c4;">Streptavidin '''</span>
-'''Investigator: '''NA, JH, LK
-'''Procedure:''' mixing of 80 µl sample with 20 µl SDS buffer and heating at 95°C for 10 min. 1 h staining, 1 night unstaining. Concentration of streptavidin was measured via UV/VIS spectroscopy.
-<math>c=\frac{A}{ℇ\ast d}\ast M\ast \mathit{Verd}.\rightarrow </math> c = 23,5 mg/ml
-'''Result: '''* 1. Lane: 8 µl Marker
-* 2. Lane: 5 µl of Streptavidin_1:10
-* 3. Lane: 5 µl of Streptavidin_1:50
-* 4. Lane: 5 µl of Pellet_1:500
-* 5. Lane: 5 µl of after 40% precipitation_1:10
-* 6. Lane: 5 µl of supernatant after 70% precipitation and centrifugation
-Successful Streptavidin production verified.
-[[Image:|top]]
+=== Chemical biotinylation of BSA ===
+'''Investigator: Niklas'''
+'''Procedure:'''
-<span style="color:#2e74b5;">'''Sequencing of P41'''</span>
+* BSA was chemically biotinylated with a 20x and 40x molar excess:
+* 10 ml of 100 mM borate buffer with 50 mM NaCl (pH 8.85)
-'''Investigator:''' NA
+* dissolve BSA (10 mg/ml)
-'''Aim of the experiment:''' Sequencing of P41
+* Add biotin-NHS-ester: 20,5 mg for 40x molar excess
-'''Procedure:'''
+* reaction over night
+</div>
-Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (100 ng/ µl) and 2 µL sequencing primer (VF2))
+=='''Tuesday, June 28th'''==
+</div>
+=='''Wednesday, June 29th'''==
+</div>
-The plasmids prepared received the following barcode:* P41 : FR11326633
+<!--- this closes the week -->
+</div>
+<!--- ^^^^ this closes the week -->
+<!--- PLEASE DO NOT TOUCH !!!! -->
+<!--- PLEASE DO NOT TOUCH !!!! -->

Difference between revisions of "Team:LMU-TUM Munich/Labjournal"

Revision as of 16:52, 29 June 2016

Contents

Labjournal

Samples

Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Sequencing of RFP-Generator (RFC25, pSB1C3)

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)

Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10

Transformation of E. coli XL1 blue with

Week 1, May 16th - May 22nd

Monday, May 16th

Streptavidin plasmids control

Streptavidin expression_trafo BL21

Tuesday, May 17th

SDS Gel Analysis

Minipreps pSb1C3-AviTag, -A3C5, pASK75-(SA1), -(SAm1)

Inoculation of pre-culture with BL21 (pASK75 (SA1)) in LB-medium

Wednesday, May 18th

Repetition of analytical gel of pSb1C3-AviTag, -A3C5

Inoculation of BL21 (pASK75 (SA1)) culture in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline

Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium

Dialysis of eGFP

MiniPrep of quickchanged pNGAL146-A2

Sequencing of P14, P15 & P19

Digestion of P16, P17, P18 & P19 with AgeI & HindIII + analytical gel

Thursday, May 19th

Re-Sequencing of P19

Cloning of A3C5 and Avi-Tag into pSB1C3

Streptavidin refolding

Biotinylation of BSA

Week 2 (May 23rd - May 29th

Week 3 (May 30th - June 5th

Week 4 (June 6th - June 12th)

Week 5 (June 13th - June 19th)

Week 6 (June 20th - June 26th)

Thursday, June 23rd

Miniprep of E. coli Xl1-Blue transformed with ligation product P80/81 (mRuby3 K1/2), P82/83 (EspP K1/2), P84/85 (StrepTag K1/2) and Trafo of K157001

Friday, June 24th

Analytical digestion and gelelectrophoresis of P83 (EspP K2) and P83 (StrepTag K2)

Sequencing of P80(mRuby3 K1), P83 (EspP K2) and P85 (StrepTag K2)

Transformation of E. coli XL1 blue with F64 (quickchanged P3(pSAm1))

Gelextraction of F67(BirA), F68(mRuby), F69(EGFR-TMD), F70(pSB1C3) and F71(pSB1C3)

Saturday, June 25th

Miniprep of E. coli Xl1-Blue transformed with P60 (mRuby/EGFR),F58 (Ligation pASK75 + Streptactin), F65 (CMV + CD4), F66 (CMV + EGFR), P70 (Short Linker)

Analytical digestion and gelelectrophoresis of F64 (quickchanged P3) for verification of succesful quickchange

Inoculation of colonies from Ligation of F69 + F70 (EGFR-TMD in pSB1C3)and F44 + F30 (mRuby in pSB1C3)

Sunday, June 26th

Miniprep of E. coli Xl1-Blue transformed with ligation product F44 + F30 (mRuby3 in pSB1C3), F66 + F70 (CMV+EGFR in pSB1C3)

Repetition of Quick-Change PCR of P3 (pASK + SAm1)

Transformation of E. coli XL1 blue with P94 (quickchanged P3(pSAm1))

Analytical digestion and gelelectrophoresis of P92 (mRuby), P93 (CMV + EGFR) and P94 (pASK75)

Week 7 (June 27th - July 3rd)

Monday, June 27th

Sequencing of P67 (EGFR-Signalpeptid)

Repetition of Quick-Change PCR of P3 (pASK + SAm1)

PCR of Genesynthesis 3 and 4

Analytical digestion and gelelectrophoresis of P88 , P89 and P90

Ligation of F67 and F71, Transformation of E. coli XL1 blue afterwards

Digestion of PCR on genesynthesis 3 and 4, and pSB1C3

Analytical digestion and gelelectrophoresis of P80 , P78 and P85

Ligation of F75 (mRuby) and F76 (NanoLuc) into F74 (pSB1C3 with Strep-Tag) and Transformation into E. coli XL1 blue

Chemical biotinylation of BSA

Tuesday, June 28th

Wednesday, June 29th