Difference between revisions of "Team:Paris Saclay/Notebook/August/25"
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====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB==== | ====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB==== | ||
| − | ''Mahnaz'' | + | ''By Mahnaz'' |
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|protocol]] of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C. | Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|protocol]] of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C. | ||
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|362 | |362 | ||
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[[File:T--Paris_Saclay--20160825_PCR_gel_Nm_Frb.jpg|400px|thumb|right|We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying]] | [[File:T--Paris_Saclay--20160825_PCR_gel_Nm_Frb.jpg|400px|thumb|right|We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying]] | ||
| + | ====Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004==== | ||
| + | ''By mathilde'' | ||
| + | |||
| + | Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol : | ||
| + | |||
| + | * 10µL of Q5 buffer | ||
| + | * 1µL of dNTPs (10mM) | ||
| + | * Primers mix (1µL each) | ||
| + | * 1µL of ligation product | ||
| + | * 0,25 µL of Q5 high fidelity polymerase | ||
| + | * 37,75 µL of nuclease free water | ||
| + | |||
| + | The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2. | ||
| + | The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension. | ||
| + | |||
| + | |||
| + | ====Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004==== | ||
| + | PCR products were put to migration following the usual protocol. | ||
| + | |||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !PCR products | ||
| + | !Expected band size (bp) | ||
| + | |- | ||
| + | |pPS16_001-pPS16_002 | ||
| + | |1920 | ||
| + | |- | ||
| + | |pPS16_003-pPS16_004 | ||
| + | |1831 | ||
| + | |} | ||
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]] | [[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]] | ||
| + | Only pPS16_001-pPS16_002 ligation product shows strands at the expected size. | ||
| + | |||
| + | ====Ligation of pPS16_003-pPS16_004==== | ||
| + | ''By Mathilde'' | ||
| + | |||
| + | * 4 µL of pPS16_003 plasmid | ||
| + | * 4 µL of pPS16_004 plasmid | ||
| + | * 1µL of T4 Buffer 10X | ||
| + | * 1µL of T4 Ligase | ||
| + | The solution is put in incubation at 4°c over night. | ||
| + | |||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Revision as of 08:11, 15 October 2016
