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Revision as of 08:06, 1 July 2016

Labjournal

Display:
General
Streptavidin
Linkers
Receptor
Optogenetics
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Week 0 sample
Week 1 02.05-08.05
Week 2 09.05-15.05
Week 3 16.05-22.05
Week 4 23.05-29.05
Week 5 30.05-05.06
Week 6 06.06-12.06
Week 7 13.06-19.06

1 kbp GeneRuler:



100 bp GeneRuler:



PageRuler Plus:


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

Muc16 P82-85 EcoRI.png 500px

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.
  • 1 µl of DNA was added to 50 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) and incubated over night at 37 °C.

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:

Muc16 Quickchange NA.JPG

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

Muc16 P88-90 NA.JPG

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

Muc16 P78-P80 2806.jpeg
Muc16 P85 2806.jpeg

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

Tuesday, June 28th

Preparative digestion and gelelectrophoresis of P83 (EspP) and P29 (DoubleTerminator)

Investigator: Julian

Aim of experiment: Preparative digestion and gelelectrophoresis of P83 (EspP) and P29 (DoubleTerminator)

Procedure:

  • Batches for analytical digestions:
  • P83: EcoRI and SpeI
  • P29: EcoRI and XbaI
volume reagent
6.5/13  µl Plasmid DNA (P83/P29)
35.5/29  µl ddH2O (P83/P29)
5 µl CutSmart buffer (10x)
1.5 µl each enzyme
50 µl TOTAL
  • Preparative gelelectrophoresis was performed at 80 V for 80 min

Muc16 P83P29 2806.jpeg

Gelelectrophoresis of F73 (pSB1C3 digst. EcoRI, AgeI)

Investigator: Julian

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

Procedure: Preparative gelelectrophoresis was performed at 100 V for 45 min using a pocket spanning over the full width of the 1% agarose gel

Muc16 F71 2806.jpeg

Wednesday, June 29th

Transformation of F83 (EspP-Fragment in Double Terminator + pSB1C3), F84 (GSY3 + pSB1C3) F85 (GSY4 + pSB1C3)

Investigator: Niklas

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 7 µl of DNA was added to 50 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 suspensions were plated on Cam-plates (inclusive rescue plate) and incubated over night at 37 °C.

Thursday, June 30th

Transformation of new E. coli XL1-blue (P75), additional plating of untransformated XL1-blue

Aim of experiment: Quality assurance

Investigator: Niklas

Procedure:

  • the new competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1 µl of DNA was added to 50 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 Cam-plates (inclusive rescue plate) and incubated over night at 37 °C.
  • additional plating of untransformated XL1-blue on Cam-, Amp and Kan-plates

Result:

Thursday, June 30th

Transformation of new E. coli XL1-blue (P75), additional plating of untransformated XL1-blue

Aim of experiment: Quality assurance

Investigator: Niklas

Procedure:

  • the new competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1 µl of DNA was added to 50 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 Cam-plates (inclusive rescue plate) and incubated over night at 37 °C.
  • additional plating of untransformated XL1-blue on Cam-, Amp and Kan-plates

Result:

Thursday, June 30th

Transformation of new E. coli XL1-blue (P75), additional plating of untransformated XL1-blue

Aim of experiment: Quality assurance

Investigator: Niklas

Procedure:

  • the new competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1 µl of DNA was added to 50 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 Cam-plates (inclusive rescue plate) and incubated over night at 37 °C.
  • additional plating of untransformated XL1-blue on Cam-, Amp and Kan-plates

Result:

Thursday, June 30th

Transformation of new E. coli XL1-blue (P75), additional plating of untransformated XL1-blue

Aim of experiment: Quality assurance

Investigator: Niklas

Procedure:

  • the new competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1 µl of DNA was added to 50 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 Cam-plates (inclusive rescue plate) and incubated over night at 37 °C.
  • additional plating of untransformated XL1-blue on Cam-, Amp and Kan-plates

Result:

Thursday, June 30th

Transformation of new E. coli XL1-blue (P75), additional plating of untransformated XL1-blue

Aim of experiment: Quality assurance

Investigator: Niklas

Procedure:

  • the new competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 1 µl of DNA was added to 50 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 Cam-plates (inclusive rescue plate) and incubated over night at 37 °C.
  • additional plating of untransformated XL1-blue on Cam-, Amp and Kan-plates

Result:

Frisday, June 30th

Transformation of P9 (pASK with Streptavidin WT) and QC-PCR product into new E. coli XL1-blue

Aim of experiment: Tranforming pASK without NdeI-cutting point into E.coli (two batches from diffrent approches were tested). And Transforming pASK_SA1 into E.coli XL-1-blue.

Investigator: Luisa

Procedure:

  • the new 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 50 µl of competent cells and gently mixed.
  • 15 min incubation on ice
  • 30 sec. heat shock at 42 °C
  • Adding of 950 µl LB-medium to each tube. 1h hour incubation at 37°C.
  • The cells were plated out on Amp-plates (inclusive rescue plate) and incubated over the weekend at 30 °C.