Difference between revisions of "Team:Paris Saclay/Notebook/July/25"

(→‎Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002)
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!'''Band Size (bp)'''
 
!'''Band Size (bp)'''
 
|960
 
|960
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|}
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====High fidelity PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001]],
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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]]====
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''By Alice''
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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.
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PCR products expected were :
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{| class="wikitable"
 +
|-
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!Plasmids
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!gBlocks
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!Primer Forward
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!Primer Reverse
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!Band size (bp)
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|-
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|pPS16_001
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|1.1
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|iPS138
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|iPS120
 +
|960
 +
|-
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|pPS16_005
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|3.1
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|iPS138
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|iPS126
 +
|960
 +
|-
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|pPS16_009
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|GFP 1-9
 +
|iPS138
 +
|iPS139
 +
|862
 
|}
 
|}

Revision as of 14:18, 25 July 2016

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
Band Size (bp) 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