Line 98:
Line 98: For each 20μl of reaction, mix the following reagents :
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.
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Latest revision as of 12:39, 17 October 2016
Monday 5th September Lab work Visualization fragments 1.1 with 1.2 Ligation AND 2.1 with 2.2 Ligation By Mahnaz
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
4µM of Fragment 1.2 PCR product from 02/09/2016
1µL of Buffer T4 10X
1µL of ligase T4 enzyme 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
4µM of Fragment 2.2 PCR product from 02/09/2016
1µL of Buffer T4 10X
1µL of ligase T4 enzyme 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 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:
Step
Temperature
Time
Initial denaturation
98°C
30sec
30 cycles
98°C
10sec
Tm
20sec
72°C
t
Final Extension
72°C
2min
Hold
4°C
infinity
Primers used were:
Matrix
Fragment 1
Fragment 2
Primers
iPS122 and iPS140
iPS123 and iPS84
Tm
72°C
72°C
t
1 min
1 min
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.
