Difference between revisions of "Team:Paris Saclay/Notebook/September/13"
(→Colony PCR of 16 clones containing GFP 1.9 in pSB1C3) |
m |
||
| (22 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
| + | {{Team:Paris_Saclay/notebook_header}} | ||
| + | |||
= Tuesday 13<sup>th</sup> September= | = Tuesday 13<sup>th</sup> September= | ||
==Lab work== | ==Lab work== | ||
===Visualization=== | ===Visualization=== | ||
| − | ====Colony PCR of 16 clones containing GFP 1.9 in pSB1C3==== | + | ====Colony PCR of 16 clones containing GFP 1.9 in pSB1C3 (pPS16_020)==== |
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
| − | A colony PCR was done for 16 clones from the 12th September. For that purpose, 16 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. | + | A colony PCR was done for 16 clones from the 12th September. For that purpose, 16 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, | + | For each clones contained in 20 μl water, 5.13 μL of the following mix were added : |
* 2.5 µL DreamTaq Buffer | * 2.5 µL DreamTaq Buffer | ||
* 0.5 µL of dNTPs (10mM) | * 0.5 µL of dNTPs (10mM) | ||
| Line 29: | Line 31: | ||
|30 sec | |30 sec | ||
|- | |- | ||
| − | | | + | |72°C |
|30 sec | |30 sec | ||
|- | |- | ||
|72°C | |72°C | ||
| − | | | + | |30sec |
|- | |- | ||
|Final Extension | |Final Extension | ||
| Line 41: | Line 43: | ||
|Hold | |Hold | ||
|4°C | |4°C | ||
| − | |$\ | + | |$\infty$ |
|} | |} | ||
| − | + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | |
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
!Matrix | !Matrix | ||
| − | !Clones containing | + | !Clones containing GFP 1.9 in pSB1C3 (pPS16_020) |
|- | |- | ||
|Primers | |Primers | ||
| − | | | + | |iPS84 and iPS140 |
|- | |- | ||
|} | |} | ||
| Line 66: | Line 68: | ||
|- | |- | ||
|GFP 1.9 in pSB1C3 | |GFP 1.9 in pSB1C3 | ||
| − | | | + | |862 |
|} | |} | ||
| − | + | [[File:T--Paris Saclay--Gel1111.png|400px|thumb|center|Result of the migration]] | |
All PCR products were at the good size. Clones 2, 7, 8 and 12 were selected and were grown at 37°C overnight. | All PCR products were at the good size. Clones 2, 7, 8 and 12 were selected and were grown at 37°C overnight. | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
====gblock 1.1 - 1.2 and gblock 2.1 - 2.2 Ligation==== | ====gblock 1.1 - 1.2 and gblock 2.1 - 2.2 Ligation==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Mahnaz'' |
A new ligation was run due to strange results obtained the 12th September. | A new ligation was run due to strange results obtained the 12th September. | ||
| − | gBlocks | + | gBlocks pPS16_001 and pPS16_002 were ligated together as following : |
| − | * 1 µL of | + | * 1 µL of pPS16_001 PCR product from 9th September |
| − | * 1 µL of | + | * 1 µL of pPS16_002 PCR product from 9th September |
* 2 µL of Buffer T4 10X | * 2 µL of Buffer T4 10X | ||
* 2 µL of ligase T4 enzyme | * 2 µL of ligase T4 enzyme | ||
| Line 111: | Line 89: | ||
gBlocks pPS16_003 and pPS16_004 were ligated together as following : | gBlocks pPS16_003 and pPS16_004 were ligated together as following : | ||
| − | * 1 µL of | + | * 1 µL of pPS16_003 PCR product from 9th September |
* 1 µL of pPS16_004 PCR product from 9th September | * 1 µL of pPS16_004 PCR product from 9th September | ||
* 2 µL of Buffer T4 10X | * 2 µL of Buffer T4 10X | ||
| Line 120: | Line 98: | ||
The ligation product was put at rooming temperature for 4 hours. | The ligation product was put at rooming temperature for 4 hours. | ||
| − | + | {{Team:Paris_Saclay/notebook_footer}} | |
Latest revision as of 10:49, 2 October 2016
