Difference between revisions of "Team:Paris Saclay/Notebook/October/2"
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=Sunday 2<sup>nd</sup> October= | =Sunday 2<sup>nd</sup> October= | ||
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===Visualization=== | ===Visualization=== | ||
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| + | ====Digestion of GFP 1.9 PCR product from the 9th September==== | ||
| + | ''By Maxence & Victor'' | ||
| + | |||
| + | As we had several issues to clones GFP 1.9 in pSB1C3 by Gibson and digestion-ligation, GFP 1.9 PCR product from the 9th September was digested by restriction NdeI enzymes in order to verify if the template we used was the good one. | ||
| + | |||
| + | For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes NdeI as following: | ||
| + | |||
| + | * 2 µL of GFP 1.9 PCR product from the 9th September | ||
| + | * 1 µL of buffer orange | ||
| + | * 1 µL of restriction enzyme NdeI | ||
| + | * 6 µL of water | ||
| + | |||
| + | The mix was incubated for 30 minutes at 37°C. Then, 20 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. | ||
====Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation==== | ====Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation==== | ||
''By Maxence & Victor'' | ''By Maxence & Victor'' | ||
| − | + | Once template and vector were cut, they were mix together with water and were precipitated by ethanol as they were not concentrated enough. The ethanol precipitation was run as following: | |
| − | + | * 28 µL of template (pPS16_019 treated by XbaI & PstI) | |
| + | * 13 µL of vector (pPS16_018 treated by SpeI & PstI) | ||
| + | * 9 µL of water | ||
| + | * 50 µL of isopropanol | ||
| + | * 5 µL of CH3COONa 3M | ||
| + | |||
| + | The mix was put 30 minutes at -20°C and was then centrifugated 10 minutes at 11 000 rpm and 4°C. The supernatant was removed and the pellet was washed twice with 100 µL of EtOH 70%. Finaly, the pullet was dried. | ||
| + | |||
| + | DNA ligase was used to join the sticky ends of the template and vector together: | ||
* 1.5 µL of Buffer T4 10X | * 1.5 µL of Buffer T4 10X | ||
| − | |||
* 1 µL of ligase T4 enzyme | * 1 µL of ligase T4 enzyme | ||
* 12.5 µL of water | * 12.5 µL of water | ||
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====Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation==== | ====Cloning of GFP 1.9 from pUC19 (pPS16_009) in pSB1C3 by digestion-ligation==== | ||
| − | ''By Maxence & Victor' | + | ''By Maxence & Victor'' |
Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together: | Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together: | ||
* 6 µL of template (pPS16_009 treated by PstI & XbaI) | * 6 µL of template (pPS16_009 treated by PstI & XbaI) | ||
| − | * 3 µL of vector ( | + | * 3 µL of vector (BbaB0015 treated by PstI & XbaI) |
* 1.5 µL of Buffer T4 10X | * 1.5 µL of Buffer T4 10X | ||
* 1 µL of ligase T4 enzyme | * 1 µL of ligase T4 enzyme | ||
* 3.5 µL of water | * 3.5 µL of water | ||
| − | The mix were incubated for 30 minutes at rooming temperature. | + | A control was done without template. The mix were incubated for 30 minutes at rooming temperature. |
| + | |||
| + | ====Gel of digestion and cleaned-up products==== | ||
| + | ''By Maxence & Victor'' | ||
| + | |||
| + | [[File:T--Paris Saclay--Gel11111115.png|400px|thumb|center|Result of the migration]] | ||
| + | |||
| + | ====Transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation==== | ||
| + | ''By Maxence & Victor'' | ||
| + | |||
| + | Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021), and with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | ||
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| + | ====Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1==== | ||
| + | "By Maxence & Victor" | ||
| + | |||
| + | The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit: | ||
| + | *pPS16_018 (FKBP - GFP 10) clone 6 | ||
| + | *pPS16_019 (FRB - GFP 11) clone 4 | ||
| + | *pPS16_009 (GFP 1.9) clone 1 | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 15:52, 14 October 2016
