Difference between revisions of "Team:Paris Saclay/Notebook/September/23"
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= Friday 23<sup>rd</sup> September= | = Friday 23<sup>rd</sup> September= | ||
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===Visualization=== | ===Visualization=== | ||
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| − | ====Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI==== | + | As gblock 2.2 and FKBP were too concentrated, they were diluated at 1/10. |
| − | ''By Maxence'' | + | |
| + | ====Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI and Gibson of PCR product containing FKBP - GFP 10 from the 22nd September x pSB1C3 treated by DpnI ==== | ||
| + | ''By Maxence, Manhaz & Coline'' | ||
Gibson was performed with cleaned up PCR products FKBP (insert 1) (obtained the 8th September) x gblock 2.2 (insert 2) (obtained the 8th September) x pSB1C3 treated by DpnI (plasmid) with the following protocol: | Gibson was performed with cleaned up PCR products FKBP (insert 1) (obtained the 8th September) x gblock 2.2 (insert 2) (obtained the 8th September) x pSB1C3 treated by DpnI (plasmid) with the following protocol: | ||
For each 20μl of reaction, mix the following reagents : | For each 20μl of reaction, mix the following reagents : | ||
| − | * | + | * 1.74 µL of insert 1 |
| − | * | + | * 1.78 µL of insert 2 |
| − | * | + | * 1.67 µL of plasmid |
| − | * | + | * 4.81 µL of water |
| + | * 10 µL of buffer mix | ||
| + | |||
| + | Furthermore, Gibson was performed with cleaned up PCR containing FKBP - GFP 10 (insert) x pSB1C3 treated by DpnI (plasmid) with the following protocol: | ||
| + | |||
| + | For each 20μl of reaction, mix the following reagents : | ||
| + | * 1.89 µL of insert | ||
| + | * 1.67 µL of plasmid | ||
| + | * 6.44 µL of water | ||
* 10 µL of buffer mix | * 10 µL of buffer mix | ||
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Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018), or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018), or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | ||
| − | + | ==== Glycerol stocks of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)==== | |
| − | + | ''By Maxence, Mahnaz & Coline'' | |
| − | + | ||
| − | ==== Glycerol stocks of clones | + | |
| − | + | ||
The glycerol stock of the bacteria with the following plasmids were made. | The glycerol stock of the bacteria with the following plasmids were made. | ||
| − | * | + | *pPS16_017 (dCas9 ST - GFP 11) clone 2 |
| − | * | + | *pPS16_017 (dCas9 ST - GFP 11) clone 7 |
| − | * | + | *pPS16_017 (dCas9 ST - GFP 11) clone 8 |
| − | + | ||
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. | 500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. | ||
| − | ====Plasmids extraction of clones | + | ====Plasmids extraction of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)==== |
| − | + | ''By Maxence, Mahnaz & Coline'' | |
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | ||
| − | * | + | *pPS16_017 (dCas9 ST - GFP 11) clone 2 |
| − | * | + | *pPS16_017 (dCas9 ST - GFP 11) clone 7 |
| − | * | + | *pPS16_017 (dCas9 ST - GFP 11) clone 8 |
| − | + | ||
| − | ==== | + | ==== Glycerol stocks of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)==== |
| − | + | ''By Maxence, Mahnaz & Coline'' | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
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The glycerol stock of the bacteria with the following plasmids were made. | The glycerol stock of the bacteria with the following plasmids were made. | ||
| − | * | + | *pPS16_020 (GFP 1.9) clone 3 |
| − | * | + | *pPS16_020 (GFP 1.9) clone 4 |
| − | * | + | *pPS16_020 (GFP 1.9) clone 7 |
| − | * | + | *pPS16_020 (GFP 1.9) clone 8 |
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. | 500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. | ||
| − | ====Plasmids extraction of clones | + | ====Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)==== |
| − | + | ''By Maxence, Mahnaz & Coline'' | |
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | ||
| − | * | + | *pPS16_020 (GFP 1.9) clone 3 |
| − | * | + | *pPS16_020 (GFP 1.9) clone 4 |
| − | * | + | *pPS16_020 (GFP 1.9) clone 7 |
| − | * | + | *pPS16_020 (GFP 1.9) clone 8 |
| − | + | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 17:08, 9 October 2016
