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=== PCR of Genesynthesis 3 and 4 ===
 
=== PCR of Genesynthesis 3 and 4 ===

Revision as of 19:34, 27 June 2016


Labjournal

Display:
General
Streptavidin
Linkers
Receptor
Optogenetics
Expand All ...
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Jump to:
Week 1 2.5-8.5
Week 2 9.5-15.5
Week 3 16.5-22.5
Week 4 23.5-29.5
Week 5 30.5-5.6
Week 6 6.6-12.6
Week 7 13.6-19.6
1 kbp GeneRuler:

100 bp GeneRuler:

PageRuler Plus:

Week Test

Monday, April 22nd

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)

Tuesday, April 23rd

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.

Wednesday, April 24th

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 2

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

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:

Photo on Monday. 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 3

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.


Digestion of PCR on genesynthesis 3 and 4

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)

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.