Difference between revisions of "Team:Paris Saclay/Notebook/July/25"
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[[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]], [[Team:Paris_Saclay/Notebook/June/28#pPS16_008|pPS16_008]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_009|pPS16_009]]==== | [[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]], [[Team:Paris_Saclay/Notebook/June/28#pPS16_008|pPS16_008]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_009|pPS16_009]]==== | ||
''By Alice'' | ''By Alice'' | ||
| − | Plasmids pPS16_001, pPS16_005 and pPS16_009 containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]]. | + | Plasmids pPS16_001, pPS16_005 and pPS16_009 containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]]. Specific [[Team:Paris_Saclay/Experiments#primers|primers]] were used for each plasmids (table below). Annealing temperature calculated 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" | {| class="wikitable" | ||
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!Primer Forward | !Primer Forward | ||
!Primer Reverse | !Primer Reverse | ||
| + | !Band size (bp) | ||
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|iPS138 | |iPS138 | ||
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|iPS138 | |iPS138 | ||
|iPS126 | |iPS126 | ||
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|pPS16_009 | |pPS16_009 | ||
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|iPS138 | |iPS138 | ||
|iPS139 | |iPS139 | ||
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{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Revision as of 14:07, 25 July 2016
Contents
Monday 25th July
Lab work
Visualization
Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002
By Mathilde
A DreamTaq PCR was made with the transformed culture pPS16_002 re-plated the day before. The usual protocol was followeed with Tm at 57°c and 5min for the initial denaturation.
We divided up the PCR mix in 6 PCR tubes and added in each one a different clone from the transformed culture. The picked colonies were re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000).
Results : PCR products expected were :
| Plasmid | pPS16_002 |
|---|---|
| Bande Size pb | 960 |
====High fidelity PCR on bacteria transformed with pPS16_001, pPS16_005, pPS16_008 and pPS16_009==== By Alice Plasmids pPS16_001, pPS16_005 and pPS16_009 containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated 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 | gBlocks | Primer Forward | Primer Reverse | Band size (bp) |
|---|---|---|---|---|
| pPS16_001 | 1.1 | iPS138 | iPS120 | 960 |
| pPS16_005 | 3.1 | iPS138 | iPS126 | 960 |
| pPS16_009 | GFP 1-9 | iPS138 | iPS139 | 862 |
