Difference between revisions of "Team:Paris Saclay/Notebook/July/26"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
=Tuesday 26<sup>th</sup> July= | =Tuesday 26<sup>th</sup> July= | ||
| − | + | ||
===Visualization=== | ===Visualization=== | ||
====High fidelity PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001]], [[Team:Paris_Saclay/Notebook/June/28#pPS16_003|pPS16_003]], [[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_009|pPS16_009]]==== | ====High fidelity PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|pPS16_001]], [[Team:Paris_Saclay/Notebook/June/28#pPS16_003|pPS16_003]], [[Team:Paris_Saclay/Notebook/June/28#pPS16_005|pPS16_005]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_009|pPS16_009]]==== | ||
''By Alice'' | ''By Alice'' | ||
| − | PCR peformed on [[Team:Paris_Saclay/Notebook/July/25#PCR_1.1_3.1_GFP|July 25]] with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]]. Specific [[Team:Paris_Saclay/Experiments#primers|primers]] were used for each plasmids (table below). Annealing temperature calculated was 72°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put | + | PCR peformed on [[Team:Paris_Saclay/Notebook/July/25#PCR_1.1_3.1_GFP|July 25]] with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]]. Specific [[Team:Paris_Saclay/Experiments#primers|primers]] were used for each plasmids (table below). Annealing temperature calculated was 72°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put on gel. PCR products were migrated 25min at 100V. |
PCR products expected were : | PCR products expected were : | ||
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| + | [[File:T--Paris_Saclay--160726_visualization_PCR1-1_2-1_3-1_GFP.jpeg|400px|thumb|right|Results of PCR products (Gblocks 1.1, 2.1, 3.1, GFP) electrophoresis]] | ||
====Low Fidelity Dreamtaq PCR of DH5α|pPS16_002==== | ====Low Fidelity Dreamtaq PCR of DH5α|pPS16_002==== | ||
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The usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] was used with TM at 57°C. | The usual [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]] was used with TM at 57°C. | ||
| − | These 6 clones were | + | These 6 clones were spread on a Petri dish containing LB, Ampicilin, IPTG and X-Gal. |
| + | |||
| + | [[File:T--Paris_Saclay--160726_visualization_PCR1-2.jpeg|400px|thumb|right|Results of PCR products (Gblocks 1.2) electrophoresis]] | ||
| + | |||
| + | ====gBlock 1.1, 3.1 and GFP1-9 insertion in puc19==== | ||
| + | ''By Caroline'' | ||
| + | |||
| + | The gblocks were inserted once again in puc19 this time after HincII were desactivated after puc19 digestion. Indeed, we noticed that those gBlocks contain a hincII restriction sites which explain why we did not obtain PCR amplification with the specific [[Team:Paris_Saclay/Experiments#primers|primers]]. | ||
| + | To carry out this insertion, the usual [[Team:Paris_Saclay/Experiments#Ligation|protocol]] was made. | ||
| + | |||
| + | ===Biobrick Characterization=== | ||
| + | ====BL21 electrocompetent cells preparation and transformation==== | ||
| + | ''By Léa, Charlène and Sylvie'' | ||
| + | |||
| + | We did an [[Team:Paris_Saclay/Experiments#ElectroCompetent|electrotransformation]] of BL21 with two different solutions of glycerol because we suspected that our solution where the root of our problem of transformation. | ||
| + | |||
| + | As we have problem with this electrotransformation, the transformation was made twice this time: once by us and another by the I2BC staff. | ||
| + | |||
| + | iGEM team's glycerol : | ||
| + | * pcl_TAA + K1372001 (time constant equal to 5.8ms) | ||
| + | * pcl_TAG + K1372001 (time constant equal to 6,2 ms) | ||
| + | * pcl_Tq + K1372001 (time constant equal to 6 ms) | ||
| + | *K1372001 (time constant equal to 6 ms) | ||
| + | Sylvie team's glycerol: | ||
| + | * pcl_TAA + K1372001 (time constant equal to 6 ms) | ||
| + | * pcl_TAG + K1372001 (time constant equal to 6 ms) | ||
| + | |||
| + | For the third conditions with iGEM team's glycerol, cells were spread on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a Petri dish with 50µL of cells and another with 500µL of cells. 150µL of BL21|K1372001 were spread on LB + Chloramphenicol medium. | ||
| + | |||
| + | For BL21 transformed with Sylvie team's glycerol, 150µL were spread on LB + Streptomycin + Chloramphenicol medium. | ||
| + | |||
| + | Two controls were made with 100µL of BL21 which were spread on LB + Chloramphénicol or LB + Streptomycin medium. | ||
| + | |||
| + | Cells grew overnight at 37°C. | ||
| − | ==== ==== | + | ====Culture of BL21 electrocompetent cells==== |
| − | ''By'' | + | ''By Léa'' |
| + | A BL21 colony was put into 4mL of liquid LB medium, and grown at 37°C, 180 rpm overnight. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 16:08, 9 October 2016
