Hi everybody,
I tried five time to clone gRNA guides and after trying several conditions nothing works.
2 guides sequences are ordered on IDT and carrying to "sticky ends" as to insert them in a plasmid previously digested and purified on gel, then verified again to be sure the purification was good.
The 2 strands are incubated at 95C for 5 min with a ramp down to 2.5 at 0.1C/sec.
I then tried the following conditions :
A
2.5µL of 10XT4 DNA ligase buffer
1µL of annealed oligos (1uM)
1.5µL of T4 DNA ligase
100ng of digested vector
QSP 25µL H2O
or
B
1µL of 10XT4 DNA ligase buffer
1µL of annealed oligos (60x times diluted)
1µL of T4 DNA ligase
7,3ng of digested vector
QSP H2O 10µL
I tried doing the ligation reaction at RT for 2 hours or 37°C for 1h
I used 2µL of the A reaction mix to transform 50µL of DH5a homemade bacteria
or 5µL of the B reaction mix with 50µL of DH5a homemade bacteria
The bacteria were transformed by 30m on ice followed by a 45s heat shock, 2m on ice. I then added 100µL of LB and platted them on LB + Amp
I'm desperate and I don't understand what I'm doing wrong. If anyone had any hindsight that would be greatly appreciated.
W
Ok thanks. Most likely the transformation is the problem:
Tom Masi Ampiciline is a bacterio static is doesn't kill the cells, they aren't happy true, but the 1h 37°C step isn't really necessary, for bacterricide antibiotics it is though. They are competent and transform very well, although I'll try to use the 10XL gold to see if that makes a difference..I'll do the recovering step also
Thanks
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