Tuesday 19th July
Lab work
Preparation of Agarose gel for DNA electrophoresis
By Terrence and Mathilde
The usual protocol was followed to prepare 600mL of agarose gel.
Biobrick characterization
Streak BL21 bacteria on LB plate
By Charlène
BL21 from glycerol stock were streaked on LB and incubated at 37°C overnight.
K1372001 pre-culture
By Naiane
One isolated colony of K1372001 from a petri dish was placed in 2mL of LB + 3µL of Chloramphenicol (20µg/mL).
The falcon tube containing the culture was incubated at 37°C overnight.
Visualization
Ligation of gBlock 1.2, 2.2, 4.1 within pUC19
By Naiane & Charlène
gBlock 1.2, 2.2, 4.1 were inserted in pUC19.
Two controls were made :
- digested pUC19 with only water
- digested pUC19 without gBlock
Transformation of DH5α with 1.2, 2.2, 4.1
By Naiane & Charlène
HeatShock competent cells were transformed with 1.2, 2.2 and 4.1. Another control was made (cells without plasmid).
Cells were plated on LB + Ampicillin + IPTG + Xgal :
- for each gBlock, one Petri dish with 50µL of cells and another with 150µL of cells
- for each control, 100µL of cells
They were incubated at 37°C overnight.
High fidelity PCR on bacteria transformed with pPS16_005 and pPS16_006
By Alice
Plasmids pPS16_005 and pPS16_006 containing gBlocks 3.1 and 3.2 were sent to sequencing. Sequencing revealed that clones 1 transformed with pPS16_005 and clone 4 transformed with pPS16_006 had the good insert in their plasmid. In ordered to assembled both inserts, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. iPS83 and iPS126 primers were used. Annealing temperature was 72°C, and initial step was prolonged to 7 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.
PCR products expected were :
| Plasmids
|
Band size (bp)
|
| pPS16_005
|
964
|
| pPS16_006
|
964
|
File:T--Paris_Saclay--160719_Visualization_échelle.JPG
Dreamtaq PCR of transformed pPS16_004 and pPS16_007
By Terrence and Mathilde
The transformed pPS16_004 and pPS16_007 with DH5α (from 18.07.2016) were amplified on PCR with the DreamTaq polymerase following the usual protocol with Tm at 57°c and 5 min for the initial denaturation.
We divided up the PCR mix in 4 PCR tubes: 25 μL of the mix was put in each tube.
1 clone from transformed pPS16_004 plate and 3 colonies from pPS16_007 were picked were soaked into each tube and re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000).
The PCR products were put on migration on agarose gel electrophoresis. Absolutely no amplicfication was observable.
Get DNA Closer
Linearization of the DS_SPcasN and DS_TDcasN plasmids
By Caroline
The DS_SPcasN and DS_TDcasN plasmids were digested with Acc65I following the usual protocol in order to facilitated the PCR.
PCR amplification from DS_SPcasN and DS_TDcasN linearized plasmids and pZA21
By Caroline
The different parts needed for de getting DNA closer tool were amplify by high fidelity PCR using Q5 following usual protocol with a TM at 65°C for pZA21 and a TM at 66°C for the 5 first cycles and at 72°C for the last 25 ones for DS-SPcasN and DS_TDcasN. Specific primers were used to amplify. These primers will allow the Gibson assembly afterward : Ptet_R and Ptet_F for the plasmids, Link-TDdcas_F and Ter_TDdcas_R for DS_TDcasN, Tet-SPdcas_F and Link-SPdcas_R for DS_SPcasN.
