Difference between revisions of "Team:Paris Saclay/Notebook/July/26"

(→‎High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009)
(→‎High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009)
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''By Alice''
 
''By Alice''
  
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PCR peformed on [[Team:Paris_Saclay/Notebook/July/25#PCR_1.1_3.1_GFP|July 25]] with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003, that 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 peformed on [[Team:Paris_Saclay/Notebook/July/25#PCR_1.1_3.1_GFP|July 25]] with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The 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. 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 :
 
PCR products expected were :
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|iPS120
 
|iPS120
 
|960
 
|960
 +
|-
 +
|pPS16_003
 +
|2.1
 +
|iPS123
 +
|iPS124
 +
|1023
 
|-
 
|-
 
|pPS16_005
 
|pPS16_005
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|862
 
|862
 
|}
 
|}
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No PCR products were expected. We supposed that initial denaturation was too long, and caused enzyme damages.
 
  
 
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Revision as of 10:10, 26 July 2016

Tuesday 26th July

Lab work

Visualization

High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009

By Alice

PCR peformed on July 25 with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The 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. 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_003 2.1 iPS123 iPS124 1023
pPS16_005 3.1 iPS138 iPS126 960
pPS16_009 GFP 1-9 iPS138 iPS139 862

By


By






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