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Team:Paris Liliane Bettencourt/Notebook/2011/09/01/
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There where ampicylin contamination in comptetent cells. Turbocells where stricken on a plate for competent cell preparation tomorow. | There where ampicylin contamination in comptetent cells. Turbocells where stricken on a plate for competent cell preparation tomorow. | ||
| + | |||
| + | ==Danyel== | ||
| + | ===GFP amber=== | ||
| + | Quick change mutation was launched last night of: GFPmut3b (E0040) and GFP + ter with the same primers. | ||
| + | Today, PCR products were digested with DpnI FastDigest enzymes and run on gel. | ||
| + | [[File:GFP QM Gel.jpg|450px|thumb|center|Ladder_Blank_GFP+ter: control_20ng_40ng_blank_GFPmut3b control_20ng]] | ||
| + | |||
| + | ===tRNA amber suppressor & T7 amber=== | ||
| + | S81: Pveg+spoVG+tRNA+ter (S24 + S58) | ||
| + | S80: T7amber + ter | ||
| + | S24: Pveg+spoVG | ||
| + | S38: tRNA in pSB1C3 | ||
| + | S58: S38 + ter | ||
| + | |||
| + | Yesturday, S81 was ligated into pHM3 and S80 was ligated into S24. | ||
| + | Both constructs were transformed and plated, then left to grow overnight. | ||
| + | The negative control of pHM3 transformation only gave one colony for 20µL insert:vector = 6:1 | ||
| + | The negative control of S24 gave innumberable colonies. | ||
| + | Nonetheless, a PCR colony was launched for both. | ||
| + | |||
| + | Since there might be a problem with one of the two S24 glycerols, miniprep preparations were made for both. | ||
| + | Ligations of S38 in S14 and S24, as well as S58 in S14 are waiting for transformation (waiting for the new competant cells) | ||
Latest revision as of 19:31, 1 September 2011
Contents |
Cyrille
Troubleshoting K606003
The glycerol was stricked on a plate. The region where the plasmids are too concentrated loose the plasmid.
Miniprep of pHM3 from glycerol
Two miniprep of pHM3 where done.
Cloning of YFP-TetR
The strain K1 (containing LacO) was open in SP. The digestion was PCR Purified.
The pHM3 was digested in EP and gel purified, but it didn't gave any yield.
There where very few of YFP-TetR left. The digested product was not visible on the gel.
Launch again YFP-TETR culture
The glycerol YFP TetR was launched again in 3 tube for miniprep tomorow
Comptetent cell debbuging and preparation
There where ampicylin contamination in comptetent cells. Turbocells where stricken on a plate for competent cell preparation tomorow.
Danyel
GFP amber
Quick change mutation was launched last night of: GFPmut3b (E0040) and GFP + ter with the same primers. Today, PCR products were digested with DpnI FastDigest enzymes and run on gel.
tRNA amber suppressor & T7 amber
S81: Pveg+spoVG+tRNA+ter (S24 + S58) S80: T7amber + ter S24: Pveg+spoVG S38: tRNA in pSB1C3 S58: S38 + ter
Yesturday, S81 was ligated into pHM3 and S80 was ligated into S24. Both constructs were transformed and plated, then left to grow overnight. The negative control of pHM3 transformation only gave one colony for 20µL insert:vector = 6:1 The negative control of S24 gave innumberable colonies. Nonetheless, a PCR colony was launched for both.
Since there might be a problem with one of the two S24 glycerols, miniprep preparations were made for both. Ligations of S38 in S14 and S24, as well as S58 in S14 are waiting for transformation (waiting for the new competant cells)
