Difference between revisions of "Team:Paris Saclay/Notebook/August/22"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
= Monday 22<sup>th</sup> August= | = Monday 22<sup>th</sup> August= | ||
| − | + | ||
===Visualization=== | ===Visualization=== | ||
| − | ====Dream Taq PCR of | + | ====Dream Taq PCR of GFP1-9 cloned in the pSB1C3==== |
''By Mathilde'' | ''By Mathilde'' | ||
| Line 15: | Line 15: | ||
Colonies were put into sterile water, with 5 minutes of denaturation (95°C in the PCR machine) before the addition of the mix. | Colonies were put into sterile water, with 5 minutes of denaturation (95°C in the PCR machine) before the addition of the mix. | ||
Annealing temperature was 57°c. | Annealing temperature was 57°c. | ||
| − | After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were loaded into a 0,8% agarose gel and then migrated at 100V for 30min. | + | After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were loaded into a 0,8% agarose gel + BET and then migrated at 100V for 30min. |
[[File:T--Paris_Saclay--160822_gel1.JPG|400px|thumb|right|Result of the PCR]] | [[File:T--Paris_Saclay--160822_gel1.JPG|400px|thumb|right|Result of the PCR]] | ||
| − | ====Sequencing of 1.2, FKBP, FRB==== | + | ====Sequencing of 1.2, FKBP, FRB cloned in pJET plasmid==== |
''Terrence and Léa'' | ''Terrence and Léa'' | ||
| Line 67: | Line 67: | ||
''Terrence and Mathilde'' | ''Terrence and Mathilde'' | ||
| − | After amplification, 50µL of each PCR product diluted 6 | + | After amplification, 50µL of each PCR product diluted 6 times with 10 µL of the gel loading dye, and 10µL of the DNA purple ladder were put into a 0,8 agarose gel + BET and migrated at 100V for 30min. |
The extraction was carried out with the [[Team:Paris_Saclay/Experiments#DNA_extraction_from_agarose_gels_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]]. | The extraction was carried out with the [[Team:Paris_Saclay/Experiments#DNA_extraction_from_agarose_gels_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]]. | ||
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''Terrence'' | ''Terrence'' | ||
| − | + | pSB1C3 was purified following the [[Team:Paris_Saclay/Experiments##Purification_:_PCR_clean-up_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]]. | |
| − | To | + | To remove any circular plasmid pSB1C3, it was digested with the enzyme DPNI. |
Nano Drop : | Nano Drop : | ||
| Line 116: | Line 116: | ||
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====Gibson on fragment 3 and fragment 4==== | ====Gibson on fragment 3 and fragment 4==== | ||
''Terrence'' | ''Terrence'' | ||
| + | We have assembled the fragment 3 and 4 following the <html> <a href="https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol">HiFi assembly protocol</a></html>with 50ng of vector and 2 times more of insert. | ||
Here is the volume used for the Gibson : | Here is the volume used for the Gibson : | ||
{| class="wikitable" | {| class="wikitable" | ||
| Line 147: | Line 146: | ||
Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | ||
| − | ==== | + | ====GFP1-9 and psB1C3 ligation product transformation in DH5a==== |
''By Léa'' | ''By Léa'' | ||
| − | + | The GFP1-9 and pSB1C3 ligation products of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. | |
| − | Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C | + | Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n. |
| − | === Interlab Study=== | + | ===Interlab Study=== |
====Transformation into DH5a cells==== | ====Transformation into DH5a cells==== | ||
''By Léa'' | ''By Léa'' | ||
| − | Devices 2 and 3, | + | Devices 2 and 3, negative control and positive control were transformed into DH5a cells, following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. |
| − | Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C | + | Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n. |
====Device 1 culture==== | ====Device 1 culture==== | ||
''By Léa'' | ''By Léa'' | ||
| − | Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C | + | Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C o/n. |
====Cell culture==== | ====Cell culture==== | ||
Latest revision as of 16:58, 9 October 2016
