Difference between revisions of "Team:Paris Saclay/Notebook/August/25"
(→Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation) |
(→pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction) |
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| + | =Thursday 25<sup>th</sup> August= | ||
| + | ===Visualization=== | ||
| − | ==== | + | ====pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction==== |
''By Terrence'' | ''By Terrence'' | ||
| − | + | The extraction was carried out following the [[Team:Paris_Saclay/Experiments#DNA_extraction_using_the_Invitrogen_ChargeSwitch.C2.AE-Pro_Plasmid_Miniprep_Kit|usual protocol]] | |
| + | The Gibson Assembly's Product (Fragment3-4) clone 6 and pPS16_009 clone 4 extraction products were digested with the restriction enzymes XBaI and PSTI | ||
| + | ====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB==== | ||
| + | ''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. |
| + | For each clones contained in 20 μl water, 4.13 μL of the following mix were added : | ||
| + | * 2.5 µL DreamTaq Buffer | ||
| + | * 0.5 µL of dNTPs (10mM) | ||
| + | * 0.5 µL of each primer(10µM) | ||
| + | * 0.13 μl of DreamTaq Pol | ||
| − | + | PCR was performed as follow: | |
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{| class="wikitable" | {| class="wikitable" | ||
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|Initial denaturation | |Initial denaturation | ||
| − | | | + | |95°C |
| − | | | + | |3 min |
|- | |- | ||
| − | |rowspan="3"| | + | |rowspan="3"|25 cycles |
| − | | | + | |94°C |
| − | | | + | |30 sec |
|- | |- | ||
| − | | | + | |Tm |
| − | | | + | |30 sec |
|- | |- | ||
|72°C | |72°C | ||
| − | | | + | |t |
|- | |- | ||
|Final Extension | |Final Extension | ||
|72°C | |72°C | ||
| − | | | + | |5min |
|- | |- | ||
|Hold | |Hold | ||
|4°C | |4°C | ||
| − | |$\ | + | |$\infinity\$ |
|} | |} | ||
| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
| − | + | ||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| − | ! | + | !Matrix |
| − | ! | + | !plasmid pJET coding sg-Nm |
| − | ! | + | !plasmid pJET coding FRB |
|- | |- | ||
|Primers | |Primers | ||
| − | | | + | |pJET R and pJET F |
| − | | | + | |pJET R and pJET F |
| + | |- | ||
| + | |Tm | ||
| + | |60.0C | ||
| + | |60.0C | ||
| + | |- | ||
| + | |t | ||
| + | |15 sec | ||
| + | |15 sec | ||
|} | |} | ||
| − | After amplification, 3 µL of each PCR products and | + | After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. |
PCR products expected were : | PCR products expected were : | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| − | ! | + | !PCR products |
| − | ! | + | !Expected band size (bp) |
|- | |- | ||
| − | | | + | |FRB |
| − | | | + | |374 |
|- | |- | ||
| − | | | + | |sg-Nm |
| − | | | + | |362 |
|} | |} | ||
| − | [[File:T-- | + | [[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 | + | 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. | ||
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| − | |||
| − | + | ====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]] | ||
| + | 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}} | ||
Latest revision as of 11:59, 17 October 2016
