Difference between revisions of "Team:Paris Saclay/Notebook/July/19"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
=Tuesday 19<sup>th</sup> July= | =Tuesday 19<sup>th</sup> July= | ||
| − | + | ||
====Preparation of Agarose gel for DNA electrophoresis==== | ====Preparation of Agarose gel for DNA electrophoresis==== | ||
''By Terrence and Mathilde'' | ''By Terrence and Mathilde'' | ||
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BL21 from glycerol stock were streaked on LB and incubated at 37°C overnight. | BL21 from glycerol stock were streaked on LB and incubated at 37°C overnight. | ||
| − | ====K1372001 pre-culture==== | + | ====DH5α|K1372001 pre-culture==== |
''By Naiane'' | ''By Naiane'' | ||
| − | One isolated colony of K1372001 from a | + | One isolated colony of DH5α|K1372001 from a Petri dish was placed in 2mL of LB + 3µL of Chloramphenicol (20µg/mL). |
| − | The | + | The Falcon tube containing the culture was incubated at 37°C overnight. |
===Visualization=== | ===Visualization=== | ||
| Line 26: | Line 26: | ||
gBlock 1.2, 2.2, 4.1 were [[Team:Paris_Saclay/Experiments#Ligation|inserted]] in pUC19. | gBlock 1.2, 2.2, 4.1 were [[Team:Paris_Saclay/Experiments#Ligation|inserted]] in pUC19. | ||
Two controls were made : | Two controls were made : | ||
| − | *digested pUC19 with only water | + | * digested pUC19 with only water |
| − | *digested pUC19 without gBlock | + | * digested pUC19 without gBlock |
| − | ====Transformation of DH5α with 1.2, 2.2, 4.1==== | + | ====Transformation of DH5α with 1.2, 2.2, 4.1<div id ="transformation__4.1_2.2_1.2"></div>==== |
''By Naiane & Charlène'' | ''By Naiane & Charlène'' | ||
| − | + | Heat-shock competent cells were [[Team:Paris_Saclay/Experiments#heat-shocktransformation|transformed]] with pPS16_002, pPS16_004 and pPS16_007. A control was also made (cells without plasmid). | |
| − | Cells were | + | Cells were spread on LB + Ampicillin + IPTG + X-Gal Petri dishe: |
| − | * for each | + | * for each plasmid, one Petri dish with 50µL of cells and another with 150µL of cells |
* 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. | ||
| Line 41: | Line 41: | ||
''By Alice'' | ''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 | + | 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 order 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. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put on gel. PCR products were migrated 25min at 100V. |
PCR products expected were : | PCR products expected were : | ||
| Line 57: | Line 57: | ||
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| − | [[File:T--Paris_Saclay--160719_Visualization_échelle.JPG| | + | [[File:T--Paris_Saclay--160719_Visualization_échelle.JPG|300px|thumb|right|Migration of pPS16_005(3.1) and pPS16_006(3.2)]] |
| − | ====Dreamtaq PCR of | + | ====Dreamtaq PCR of DH5α|pPS16_004 and DH5α|pPS16_007 ==== |
''By Terrence and Mathilde'' | ''By Terrence and Mathilde'' | ||
| − | + | DH5α|pPS16_004 and DH5α|pPS16_007 ([[Team:Paris_Saclay/Notebook/July/18#Visualization|from 18.07.2016)]] were amplified on PCR with the DreamTaq polymerase following the usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] with Tm at 57°C. | |
| − | We divided | + | We divided the PCR mix in 4 PCR tubes: 25 μL of the mix was put in each tube. |
| − | 1 clone from | + | 1 clone from DH5α|pPS16_004 plate and 3 colonies from DH5α|pPS16_007 were harvested and inoculated into each tube and spread on Petri dish with LB + Ampicillin (50µg/mL) + X-Gal/IPTG (1/1000). |
| − | The PCR products were | + | The PCR products were migrated on an agarose gel. No amplification was seen. |
| − | === | + | ===Bringging DNA Closer=== |
====Linearization of the DS_SPcasN and DS_TDcasN plasmids==== | ====Linearization of the DS_SPcasN and DS_TDcasN plasmids==== | ||
''By Caroline'' | ''By Caroline'' | ||
| − | The DS_SPcasN and DS_TDcasN plasmids were digested with Acc65I following the [[Team:Paris_Saclay/Experiments#PlasmidDigestion|usual]] protocol in order to facilitated the PCR. | + | The DS_SPcasN- and DS_TDcasN- plasmids were digested with Acc65I following the [[Team:Paris_Saclay/Experiments#PlasmidDigestion|usual]] protocol in order to facilitated the PCR. |
| − | ====PCR amplification from | + | ====PCR amplification from DS-SPcasN- and DS-TDcasN- linearized plasmids and pZA21==== |
''By Caroline'' | ''By Caroline'' | ||
| − | The different parts needed for | + | The different parts needed for the bring DNA closer tool were amplify by high fidelity PCR using Q5 following [[Team:Paris_Saclay/Experiments#Q5PCR|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-. [[Team:Paris_Saclay/Experiments#primers|Specific primers]] were used to amplify. These primers will allow 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-. |
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 15:39, 9 October 2016
