Difference between revisions of "Team:Paris Saclay/Notebook/August/26"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
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= Friday 26<sup>th</sup> August= | = Friday 26<sup>th</sup> August= | ||
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===Visualization=== | ===Visualization=== | ||
| + | ====Purification of pPS16-003 and pPS16_004 ligation products==== | ||
| + | ''By Mathilde'' | ||
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| + | Ligation products pPS16-003 and pPS16_004 frome the [[Team:Paris_Saclay/Notebook/August/25#Visualization|day before]] were purificated according to the usual [[Team:Paris_Saclay/Experiments#Purification_:_PCR_clean-up_with_NucleoSpin_Gel_and_PCR_Clean-up|protocol]] | ||
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====Q5 high fidelity PCR of pPS16_003 - pPS16_004 ligation product==== | ====Q5 high fidelity PCR of pPS16_003 - pPS16_004 ligation product==== | ||
'' By Mathilde '' | '' By Mathilde '' | ||
The Q5 PCR was conducted following the exact same protocol than the [[Team:Paris_Saclay/Notebook/August/25#Visualization|25/08/2016]] on the ligation product from the day before. | The Q5 PCR was conducted following the exact same protocol than the [[Team:Paris_Saclay/Notebook/August/25#Visualization|25/08/2016]] on the ligation product from the day before. | ||
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| + | [[File:T--Paris_Saclay--160826_gel_ligation_fragm2.png|400px|thumb|centre|Migration Results]] | ||
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| + | The two bands are at the expected size (1860 pb), so the fragment 2 (16-003 and pPS16_004 ligation product) is purified. | ||
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| + | ==== Purification of fragments 1(16-001 + pPS16_002) and 2 (16-003 + pPS16_004)==== | ||
| + | '' By Mathilde'' | ||
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| + | The purification was made following the usual [[Team:Paris_Saclay/Experiments#Purification_:_PCR_clean-up_with_NucleoSpin_Gel_and_PCR_Clean-up|protocol]]. | ||
| + | Each of those purification products were quantified on nanodrop. | ||
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| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Segment | ||
| + | !DNA quantity (ng/µL) | ||
| + | !260/230 | ||
| + | !260/280 | ||
| + | |- | ||
| + | |1 | ||
| + | |84,20 | ||
| + | |1,25 | ||
| + | |1,84 | ||
| + | |- | ||
| + | |2 | ||
| + | |169,88 | ||
| + | |1,29 | ||
| + | |1,83 | ||
| + | |} | ||
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| + | Enough DNA quantity was obtained to perform a Gibson assembly on segments 1 and 2. | ||
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| + | ====Gibson Assembly of segments 1 and 2==== | ||
| + | ''By mathilde'' | ||
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| + | Two preparations were made : | ||
| + | * the 1-2 Gibson assembly product tube : 1,12µL of purified segment 1 and 0,56µL of purified segment 2 + 0,85µL of digested pSB1C3 + 7,47 µL of sterile water + 10µL of NEB Builder Hifi DNA Assembler Master Mix | ||
| + | * the control tube : 0,85µL of digested pSB1C3 + 17,47 µL of sterile water | ||
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| + | Gibson products were incubated 1h at 52°c. | ||
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| + | ====Transformation in DH5α with the 1-2 Gibson assembly product==== | ||
| + | ''By Mathilde'' | ||
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| + | The transformation was performed following the usual [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|protocol]] with 2µL of plasmid. | ||
| + | * 50, 150 and 350µL of the transformed DH5α with the 1-2 Gibson assembly product were plated on 3 plates of LB + chloramphenicol (30µg/mL) | ||
| + | * 350 µL of each Gibson assembly control and transformation control were plated on 2 plates of B + chloramphenicol (30µg/mL) | ||
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| + | Petri cultures were incubated at 37°c overnight. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 17:00, 9 October 2016
