Difference between revisions of "Team:Paris Saclay/Notebook/August/18"

(→‎pPS16_002, pPS16_010, pPS16_013 and pPS16_014 PCR with pJET)
(→‎Purification of gel bands 1.2 (gBlock) and 4 (ligation))
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Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mix in a tube. The mix was incubated 1 hour at room temperature.
 
Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mix in a tube. The mix was incubated 1 hour at room temperature.
  
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====Purification of gel bands 1.2 (gBlock) and 4 (ligation)====
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====PCR Clean -up of gel bands 1.2 (gBlock) and 4 (ligation)====
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The gBlock 1.2 which was extracted yesterday and ligation of the assembled gBlock 4 were purified following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]].
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''By Naiane''
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The bands 1.2 and 4 excised yesterday were used to continue the NucleoSpin Gel and PCR Clean-up kit[[Team:Paris_Saclay/Experiments#Purification|protocol]].
  
 
==== pPS16_002, pPS16_010, pPS16_013 and pPS16_014 PCR with pJET====
 
==== pPS16_002, pPS16_010, pPS16_013 and pPS16_014 PCR with pJET====

Revision as of 15:03, 18 August 2016

Thursday 18th August

Lab work

Visualization

Fragment 3 digestion with Eco47III

By Alice

gBlocks 3 were digested with eco47III enzyme following this protocol. The mix was incubated 2 hours at 37°C. Then eco47III was inactivated during 20 min at 60°C. Then digestion products were purified following this protocol.

Digested fragment 3 ligation with fragment 4

By Alice

Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mix in a tube. The mix was incubated 1 hour at room temperature.

PCR Clean -up of gel bands 1.2 (gBlock) and 4 (ligation)

By Naiane The bands 1.2 and 4 excised yesterday were used to continue the NucleoSpin Gel and PCR Clean-up kitprotocol.

pPS16_002, pPS16_010, pPS16_013 and pPS16_014 PCR with pJET

By Léa and Terrence

Dream Taq PCR were performed on 6 colonies of DH5a transformed on the 17/08/2016 using pJET-FRB /FKBP / Nm. 12 colonies were plated on Petri dishes and inoculated into tubes containing 3mL of LB agar and amp.

For DH5a pJET / 1.2, 12 colonies were screened, plated and inoculated into tubes. The PCR was performed using the usual protocol with universal primers pJET.

Lysis at 95°C for 5 min and Tm = 52°C

The PCR were put onto agarose gel for migration.

Biobrick characterization

B-Galactosidase and luciferase test on transformed BL21

By Charlene and Mathilde

Cultures were spun down for 5min at 13000rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rpm at 4°C.

For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured.