Line 34:
Line 34: * for each control, 100µL of cells
* for each control, 100µL of cells
They were incubated at 37°C overnight.
They were incubated at 37°C overnight.
+
+ ====High fidelity PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_006|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 [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]]. [[Team:Paris_Saclay/Experiments#primers|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 :
+
+ {| class="wikitable"
+ |-
+ !Plasmids
+ !Band size (bp)
+ |-
+ |pPS16_005
+ |964
+ |-
+ |pPS16_006
+ |964
+ |}
===Get DNA Closer===
===Get DNA Closer===
Revision as of 15:36, 19 July 2016
Tuesday 19th July Lab work 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
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.
