Difference between revisions of "Team:Paris Saclay/Notebook/October/3"
(→Colony PCR of X clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)) |
(→Monday 3rd October) |
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| Line 51: | Line 51: | ||
|- | |- | ||
!Matrix | !Matrix | ||
| − | !Clones containing FKBP - GFP 10 in pSB1C3 ( | + | !Clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) |
|- | |- | ||
|Primers | |Primers | ||
| Line 69: | Line 69: | ||
|FRB - GFP 11 - FKBP - GFP 10 | |FRB - GFP 11 - FKBP - GFP 10 | ||
|1714 | |1714 | ||
| + | |} | ||
| + | |||
| + | GEL | ||
| + | |||
| + | ====Colony PCR of X clones containing GFP 1.9 in pSB1C3 (pPS16_020)==== | ||
| + | ''By Maxence'' | ||
| + | |||
| + | For that purpose, X clones 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 + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C. | ||
| + | |||
| + | For each clones contained in 20 μl water, 5.13 μL of the following mix were added : | ||
| + | * 2.5 µL DreamTaq Buffer | ||
| + | * 0.5 µL of dNTPs (10mM) | ||
| + | * 1 µL of each primer mix (10µM) | ||
| + | * 0.13 μl of DreamTaq Pol | ||
| + | |||
| + | PCR was performed as follow: | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Step | ||
| + | !Temperature | ||
| + | !Time | ||
| + | |- | ||
| + | |Initial denaturation | ||
| + | |95°C | ||
| + | |3 min | ||
| + | |- | ||
| + | |rowspan="3"|30 cycles | ||
| + | |95°C | ||
| + | |30 sec | ||
| + | |- | ||
| + | |48.4°C | ||
| + | |30 sec | ||
| + | |- | ||
| + | |72°C | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Final Extension | ||
| + | |72°C | ||
| + | |7 min | ||
| + | |- | ||
| + | |Hold | ||
| + | |4°C | ||
| + | |$\infty$ | ||
| + | |} | ||
| + | |||
| + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | ||
| + | |||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Matrix | ||
| + | !Clones containing GFP 1.9 in pSB1C3 (pPS16_020) | ||
| + | |- | ||
| + | |Primers | ||
| + | |iPS168 and iPS169 | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | 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 : | ||
| + | |||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !PCR products | ||
| + | !Expected band size (bp) | ||
| + | |- | ||
| + | |GFP 1.9 | ||
| + | |1135 | ||
|} | |} | ||
Revision as of 19:39, 2 October 2016
