Difference between revisions of "Team:Paris Saclay/Notebook/October/3"
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= Monday 3<sup>rd</sup> October= | = Monday 3<sup>rd</sup> October= | ||
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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. |
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 : | ||
| Line 35: | Line 35: | ||
|- | |- | ||
|72°C | |72°C | ||
| − | | | + | |30 sec |
|- | |- | ||
|Final Extension | |Final Extension | ||
| Line 51: | Line 51: | ||
|- | |- | ||
!Matrix | !Matrix | ||
| − | !Clones containing | + | !Clones containing GFP 1.9 in pSB1C3 (pPS16_020) |
|- | |- | ||
|Primers | |Primers | ||
| Line 67: | Line 67: | ||
!Expected band size (bp) | !Expected band size (bp) | ||
|- | |- | ||
| − | | | + | |GFP 1.9 |
| − | | | + | |1135 |
|} | |} | ||
| + | [[File:T--Paris Saclay--Gel11111116.png|400px|thumb|center|Result of the migration]] | ||
| + | |||
| + | [[File:T--Paris Saclay--Gel11111117.png|400px|thumb|center|Result of the migration]] | ||
| + | |||
| + | ====Digestion of GFP 1.9 in pUC19 (pPS16_009) clone 1==== | ||
| + | ''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. | ||
| + | |||
| + | 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 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: | ||
| + | |||
| + | * 5 µL of GFP 1.9 (pPS16_009) clone 1 | ||
| + | * 2 µL of buffer orange | ||
| + | * 2 µL of restriction enzyme BsaBI | ||
| + | * 11 µL of water | ||
| + | |||
| + | 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==== | ||
| + | ''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 as following: | ||
| + | |||
| + | * 5 µL of plasmid | ||
| + | * 2 µL of buffer FD | ||
| + | * 2 µL of restriction enzyme EcoRI | ||
| + | * 11 µL of water | ||
| + | |||
| + | The mix were incubated for 15 minutes at 37°C. | ||
| + | ====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. | ||
| + | [[File:T--Paris Saclay--Gel11111118.png|400px|thumb|center|Result of the migration]] | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 15:53, 14 October 2016
