Difference between revisions of "Team:Paris Saclay/Notebook/July/8"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
| − | = | + | =Friday 8<sup>th</sup> July= |
| − | == | + | |
| + | ===Bringing DNA closer=== | ||
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| + | [[File:T--Paris_Saclay--160708_bringingDNAcloser_échelle.jpg|400px|thumb|right|Migration of SP, SP1, SP2, NM, ST1, TD]] | ||
===Visualization=== | ===Visualization=== | ||
| − | ====Colony screening PCR | + | ====Colony screening PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/July/4#pPS16_004|pPS16_004]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]]==== |
''By Alice'' | ''By Alice'' | ||
| + | <div id="Colony_screening_PCR"></div> | ||
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| + | After transformations, only white bacteria were selected (blue/white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers ([[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]]) upstream and downstream the insertion site are chosen. Annealing temperature was 53°C. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were put on gel and migrated at 100v during 30 min. | ||
| − | + | PCR products expected were : | |
{| class="wikitable" | {| class="wikitable" | ||
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|- | |- | ||
|pPS16_004 | |pPS16_004 | ||
| − | | | + | |865 |
|- | |- | ||
|pPS16_007 | |pPS16_007 | ||
| − | | | + | |763 |
|} | |} | ||
| − | No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of steril water. The same PCR will be performed on 11 | + | [[File:T--Paris_Saclay--160711_visualization2_échelle.jpg|400px|thumb|right|pPS16_004 (2.2) and pPS16_007 (4.1) Migration]] |
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| + | No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of MilliQ and steril water allowing the reduction of RNAse. The same PCR will be performed on July 11. | ||
====Electrophoresis of the screening PCR products==== | ====Electrophoresis of the screening PCR products==== | ||
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Some clones showed PCR products with lengths close to those expected. | Some clones showed PCR products with lengths close to those expected. | ||
| − | ====High fidelity PCR | + | ====High fidelity PCR<div id="high_fidelity_PCR"></div>==== |
''By Caroline'' | ''By Caroline'' | ||
| − | The clones (from [[Team:Paris_Saclay/Notebook/July/5#Transformation_5/07|05/07/2016]])found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme Q5. We used a high fidelity enzyme | + | The clones (from [[Team:Paris_Saclay/Notebook/July/5#Transformation_5/07|05/07/2016]]) found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme: Q5. We used a high fidelity enzyme to reduce the error ratio and send to sequencing the PCR products. A PCR was carried out using Q5<sup>®</sup> High-Fidelity 2X Master Mix and following the [[Team:Paris_Saclay/Experiments#Q5PCR|usual protocol]] with pUC19 primers [[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]]. |
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{| class="wikitable" | {| class="wikitable" | ||
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|746 | |746 | ||
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| + | [[File:T--Paris_Saclay--160708_visualization_échelle_pas_sure.jpg|400px|thumb|right|pPS16_001 (1.1), pPS16_002 (1.2) and pPS16_003 (2.1) Migration]] | ||
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| + | [[File:T--Paris_Saclay--160708_visualization2_échelle.jpg|400px|thumb|right|pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration]] | ||
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| + | [[File:T--Paris_Saclay--160708_visualization3_échelle.jpg|400px|thumb|right|pPS16_004 (2.2) and pPS16_007 (4.1) Migration]] | ||
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| + | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 14:49, 9 October 2016
