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= Friday 23<sup>rd</sup> September= | = Friday 23<sup>rd</sup> September= | ||
− | + | ||
===Visualization=== | ===Visualization=== | ||
+ | ====NanoDrop Measurements==== | ||
+ | ''By Maxence, Manhaz & Coline'' | ||
− | ==== | + | {| class="wikitable" |
− | + | !Sample | |
+ | !Concentration (ng/µL) | ||
+ | |- | ||
+ | |FKBP amplification product from the 8th September<div id="FKBP amplification product from the 8th September"></div> | ||
+ | |231.55 | ||
+ | |- | ||
+ | |gblock 2.2 amplification product from the 8th September<div id="gblock 2.2 amplification product from the 8th September"></div> | ||
+ | |183.9 | ||
+ | |- | ||
+ | |pSB1C3 treated by DpnI from the 15th September<div id="pSB1C3 treated by DpnI from the 15th September"></div> | ||
+ | |59.68 | ||
+ | |- | ||
+ | |PCR product containing FKBP - GFP 10 from the 22nd September<div id="PCR product containing FKBP - GFP 10 from the 22nd"></div> | ||
+ | |38.19 | ||
+ | |- | ||
+ | |} | ||
− | + | As gblock 2.2 and FKBP were too concentrated, they were diluated at 1/10. | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ====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: | |
− | + | ||
− | + | 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 | ||
− | + | Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C. | |
− | + | ||
− | + | ||
− | + | ||
− | + | ====Transformation of DH5a cells with FKBP - GFP 10 in pSB1C3 (pPS16_018) obtained by Gibson==== | |
+ | ''By Maxence & Mahnaz'' | ||
− | + | 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 | + | ==== Glycerol stocks of clones 2, 7 and 8 containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)==== |
− | + | ''By Maxence, Mahnaz & Coline'' | |
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 | + | *pPS16_017 (dCas9 ST - GFP 11) clone 2 |
− | *pPS16_017 (dCas9 ST - GFP 11) clone | + | *pPS16_017 (dCas9 ST - GFP 11) clone 7 |
− | *pPS16_017 (dCas9 ST - GFP 11) clone | + | *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 | + | *pPS16_017 (dCas9 ST - GFP 11) clone 2 |
− | *pPS16_017 (dCas9 ST - GFP 11) clone | + | *pPS16_017 (dCas9 ST - GFP 11) clone 7 |
− | *pPS16_017 (dCas9 ST - GFP 11) clone | + | *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'' | ||
+ | |||
+ | 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. | ||
+ | |||
+ | ====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: | ||
+ | *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