Difference between revisions of "Team:Paris Saclay/Notebook/September/5"
MahnaziGEM (Talk | contribs) |
MahnaziGEM (Talk | contribs) (→Gibson of cleaned up PCR products fragment 1 and fragment 2 in pSB1C3 treated by DpnI) |
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''By Mahnaz'' | ''By Mahnaz'' | ||
| − | Fragment 1.1 and 1.2 were ligated together as following | + | Fragment 1.1 and 1.2 were ligated together as following to create fragment 1 |
* 4µL of Fragment 1.1 PCR product from the 02/09/2016 | * 4µL of Fragment 1.1 PCR product from the 02/09/2016 | ||
* 4µM of Fragment 1.2 PCR product from 02/09/2016 | * 4µM of Fragment 1.2 PCR product from 02/09/2016 | ||
| Line 13: | Line 13: | ||
* 1µL of ligase T4 enzyme | * 1µL of ligase T4 enzyme | ||
| − | Fragment 2.1 and 2.2 were ligated together as following | + | Fragment 2.1 and 2.2 were ligated together as following to create fragment 2 |
* 4µL of Fragment 2.1 PCR product from the 02/09/2016 | * 4µL of Fragment 2.1 PCR product from the 02/09/2016 | ||
* 4µM of Fragment 2.2 PCR product from 02/09/2016 | * 4µM of Fragment 2.2 PCR product from 02/09/2016 | ||
| Line 20: | Line 20: | ||
The ligation product was put at room temperature for 1 hour. | The ligation product was put at room temperature for 1 hour. | ||
| + | |||
| + | ==== Clean-up of ligation products ==== | ||
| + | ''By Mahnaz'' | ||
| + | |||
| + | Ligation product (fragement 1 and fragment 2) obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. | ||
| + | |||
| + | ====Q5 PCR and phusion PCR on ligation product ==== | ||
| + | ''By Mahnaz'' | ||
| + | |||
| + | Q5 and phusion PCR was performed on the ligation product (fragment 1 and fragment 2) with the following protocol: | ||
| + | For each 50μl of reaction, mix the following reagents: | ||
| + | * 1 µL of plasmid | ||
| + | * 1 µL of dNTPs (10mM) | ||
| + | * 1 µL of each primer mix (10µM) | ||
| + | * 10 µL of Q5 buffer (5X) | ||
| + | * 0,5 µL of Q5 high fidelity polymerase | ||
| + | * 35,5 µL of nuclease free water | ||
| + | |||
| + | Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. | ||
| + | Perform PCR as follow: | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Step | ||
| + | !Temperature | ||
| + | !Time | ||
| + | |- | ||
| + | |Initial denaturation | ||
| + | |98°C | ||
| + | |30sec | ||
| + | |- | ||
| + | |rowspan="3"|30 cycles | ||
| + | |98°C | ||
| + | |10sec | ||
| + | |- | ||
| + | |Tm | ||
| + | |20sec | ||
| + | |- | ||
| + | |72°C | ||
| + | |t | ||
| + | |- | ||
| + | |Final Extension | ||
| + | |72°C | ||
| + | |2min | ||
| + | |- | ||
| + | |Hold | ||
| + | |4°C | ||
| + | |infinity | ||
| + | |} | ||
| + | [[Team:Paris_Saclay/Experiments#primers|Primers]] used were: | ||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Matrix | ||
| + | ! Fragment 1 | ||
| + | ! Fragment 2 | ||
| + | |- | ||
| + | |Primers | ||
| + | |iPS122 and iPS140 | ||
| + | |iPS123 and iPS84 | ||
| + | |- | ||
| + | |Tm | ||
| + | |72°C | ||
| + | |72°C | ||
| + | |- | ||
| + | |t | ||
| + | |1 min | ||
| + | |1 min | ||
| + | |} | ||
| + | |||
| + | [[File:T--Paris_saclay--0509.extension.png|400px|thumb|center|Result of migration ]] | ||
| + | The PCR product of ligation is not in high concentration (mostly with phusion polymerase) | ||
| + | Following Gibson assembly was done by both fragments. | ||
| + | |||
| + | ====Gibson of cleaned up PCR products fragment 1 and fragment 2 in pSB1C3 treated by DpnI==== | ||
| + | ''By Manhaz '' | ||
| + | |||
| + | Gibson was performed with cleaned up PCR products fragment1 x fragment 2 x pSB1C3 treated by DpnI (plasmid) with the following protocol: | ||
| + | |||
| + | For each 20μl of reaction, mix the following reagents : | ||
| + | |||
| + | * 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 | ||
| + | |||
| + | Gibson products were incubated 1h at 52°c. | ||
| + | |||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 12:39, 17 October 2016
