Difference between revisions of "Team:Paris Saclay/Notebook/August/17"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
= Wednesday 17<sup>th</sup> August= | = Wednesday 17<sup>th</sup> August= | ||
| − | == | + | |
| + | ===Biobrick characterisation=== | ||
| + | ====Culture of BL21|K1372001 and pcl_TAA, pclTAG or pclTq==== | ||
| + | ''By Charlène'' | ||
| + | |||
| + | 3 clones from each transformation condition (K1372001 and pcl_TAA, pclTAG or pclTq) were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL). | ||
| + | Then each solution was split into 3x150µL and 350µL of mix of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture. | ||
| + | Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM. | ||
| + | |||
===Visualization=== | ===Visualization=== | ||
| − | ==== Extraction of | + | ==== Extraction of pPS16_003, pPS16_004 and GFP1-9_PSB1C3 (clones 2 and 3) ==== |
''By Charlène'' | ''By Charlène'' | ||
| − | Clones 2 and 3 of GFP1-9_PSB1C3, and | + | Clones 2 and 3 of GFP1-9_PSB1C3, and pPS16_003, pPS16_004 were extracted with the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|Plasmid MiniPrep kit]]. |
| − | The extracts were put | + | The extracts were put on migration on a 0.8%agarose gel with BET. |
==== Purification of gBlocks 2, 3 and 4 ==== | ==== Purification of gBlocks 2, 3 and 4 ==== | ||
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A heat shock transformation was made on the cloning samples using the following [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]] | A heat shock transformation was made on the cloning samples using the following [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]] | ||
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====Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation==== | ====Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation==== | ||
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| − | Annealing temperature was 70°C | + | Annealing temperature was 70°C. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed on gel and migrated at 100v during 30 min. |
PCR products expected were : | PCR products expected were : | ||
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''By Terrence'' | ''By Terrence'' | ||
| − | A second migration on agarose gel was performed with the total volume of the PCR products | + | A second migration on agarose gel was performed with the total volume of the PCR products 1.2 and 4. |
| − | The migration allowed us to excise the DNA fragments from the agarose gel below and | + | The migration allowed us to excise the DNA fragments from the agarose gel below and carried on with the Step 1 of the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. |
| − | The | + | The excised bands from Step 1 were incubated with the buffer NT1 at 4°C overnight. |
[[File:T--Paris_Saclay--20160817_Gel_extraction_1-2_lig4.jpeg.JPG|500px|thumb|right|Gel extraction fo 1.2 and Lig 4]] | [[File:T--Paris_Saclay--20160817_Gel_extraction_1-2_lig4.jpeg.JPG|500px|thumb|right|Gel extraction fo 1.2 and Lig 4]] | ||
| − | ====Low Fidelity DreamTaqPCR of DH5a| | + | ====Low Fidelity DreamTaqPCR of DH5a|pPS16_020, DH5a|pPS16_010, DH5a|pPS16_012 and DH5a|pPS16_013==== |
''By Mathilde'' | ''By Mathilde'' | ||
| − | PCR was performed on clones 1 to 5 for | + | PCR was performed on clones 1 to 5 for pPS16_020, clones 1,2,3,5 and 6 for pPS16_010, clones 1,2,3 and 6 for pPS16_012, and for clones 1,4 and 6 for pPS16_013. Both white and blue colonies were picked for each plasmids. |
Thus, the PCR mix was done for 17 tubes following the usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]]. But 1µL of dNTPs were used instead of 2,5µL, and colonies were put in the water before the addition of the rest of the mix for each sample. | Thus, the PCR mix was done for 17 tubes following the usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]]. But 1µL of dNTPs were used instead of 2,5µL, and colonies were put in the water before the addition of the rest of the mix for each sample. | ||
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Each clone was put in liquide culture (LB 3mL + Ampicillin 50µg/mL) | Each clone was put in liquide culture (LB 3mL + Ampicillin 50µg/mL) | ||
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Each PCR product was placed on agarose gel to migrate. | Each PCR product was placed on agarose gel to migrate. | ||
| − | 10µL of violet ladder was used | + | 10µL of violet ladder was used. |
PCR products expected were : | PCR products expected were : | ||
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|- | |- | ||
!'''Plasmid''' | !'''Plasmid''' | ||
| − | ! | + | !pPS16_020 |
!pPS16_010 | !pPS16_010 | ||
!pPS16_012 | !pPS16_012 | ||
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|} | |} | ||
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| + | [[File:T--Paris_Saclay--20160811_gel_Q5_PCR_mathilde.JPG|400px|thumb|right|Result of the PCR]] | ||
No PCR product presented the expected size. | No PCR product presented the expected size. | ||
| − | |||
====Digestion of pPS16_013 clones 2 and 3==== | ====Digestion of pPS16_013 clones 2 and 3==== | ||
''By Mathilde'' | ''By Mathilde'' | ||
| − | The two plasmids were digested with the | + | The two plasmids were digested with the XbaI and PstI enzymes with the following protocol : |
* 2µL of plasmid | * 2µL of plasmid | ||
| − | * 0,5µL of each | + | * 0,5µL of each restriction enzyme |
| − | * 1µL of | + | * 1µL of Green Fast Digestion Buffer |
* 6 µL of sterile water | * 6 µL of sterile water | ||
| − | The two | + | The two preparations were put for incubation during 5 min at 37°c, and then directly put to migrate. |
| + | |||
| + | [[File:T--Paris_Saclay--20160811_gel_migration_PCR_mathilde.JPG|400px|thumb|right|Result of the migration]] | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 16:48, 9 October 2016
