Difference between revisions of "Team:Paris Saclay/Notebook/October/3"
(→Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6) |
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===Visualization=== | ===Visualization=== | ||
| − | ====Colony PCR of | + | ====Colony PCR of 32 clones containing GFP 1.9 in pSB1C3 (pPS16_020)==== |
| − | ''By Maxence'' | + | ''By Maxence & Caroline'' |
| − | For that purpose, | + | For that purpose, 32 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. |
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For each clones contained in 20 μl water, 5.13 μL of the following mix were added : | For each clones contained in 20 μl water, 5.13 μL of the following mix were added : | ||
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====Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1==== | ====Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Caroline'' |
Extracted pPS16_009 from the 2nd October was digested by several restriction enzymes in order to verify if the template we used was the good one. | Extracted pPS16_009 from the 2nd October was digested by several restriction enzymes in order to verify if the template we used was the good one. | ||
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For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following: | For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following: | ||
| − | * | + | * 5 µL of GFP 1.9 (pPS16_009) clone 1 |
* 2 µL of buffer orange | * 2 µL of buffer orange | ||
* 2 µL of restriction enzyme NdeI | * 2 µL of restriction enzyme NdeI | ||
| − | * | + | * 11 µL of water |
Furthermore, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes BsaBI as following: | Furthermore, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes BsaBI as following: | ||
| − | * | + | * 5 µL of GFP 1.9 (pPS16_009) clone 1 |
* 2 µL of buffer orange | * 2 µL of buffer orange | ||
* 2 µL of restriction enzyme BsaBI | * 2 µL of restriction enzyme BsaBI | ||
| − | * | + | * 11 µL of water |
| − | The mix were incubated for 30 minutes at 37°C. | + | The mix were incubated for 30 minutes at 37°C. |
| − | + | ||
| − | + | ||
====Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6==== | ====Digestion of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6==== | ||
| − | ''By Maxence'' | + | ''By Maxence & Caroline'' |
Extracted pPS16_019 and pPS16_018 from the 2nd October were digested by restriction enzymes EcoRI in order to verify the concentration. | Extracted pPS16_019 and pPS16_018 from the 2nd October were digested by restriction enzymes EcoRI in order to verify the concentration. | ||
| Line 171: | Line 101: | ||
For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following: | For that purpose, GFP 1.9 in pUC19 (pPS16_009) clone 1 was cut by restriction enzymes NdeI as following: | ||
| − | * | + | * 5 µL of plasmid |
* 2 µL of buffer FD | * 2 µL of buffer FD | ||
* 2 µL of restriction enzyme EcoRI | * 2 µL of restriction enzyme EcoRI | ||
| − | * | + | * 11 µL of water |
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| + | The mix were incubated for 15 minutes at 37°C. | ||
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| + | ====Gel of digested products==== | ||
| + | ''By Maxence & Caroline'' | ||
| − | + | After digestion, 20 µL of digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. | |
GEL | GEL | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Revision as of 15:53, 3 October 2016
