When I transform linear PCR products that contain the KanMX4 cassette, I usually plate on YDP, incubate overnight, and then replica plate on G418. This works a charm. However, this time, I am transforming a non-integrating plasmid with the KanMX4 cassette, and I am not getting any transformants. I was wondering whether overnight recovery might not be too long and causes the cells to lose the plasmid?
Has anybody done this type of transformation before and how long did you recover for (on plate or in liquid?)
Thank you for any feedback.
I usually use 1-2 hours for recovery in liquid YPD, overnight is a bit excessive. You need to linearize the plasmid in the homology region otherwise it will not integrate properly.
Hi Hanna. Thank you for your response. I do not want it to integrate. It is crisp plasmid. I will try your suggestion of 1-2 hours. Thank you :)
Are you cotransforming a repair template? because if you dont it will give you a very low number of colonies.
yes. First time I did do that and got nothing. Then I decided to optimise the transformation using the parental strain lacking the guide sequence and I didn't co-transform the repair templates. I still got nothing. I think my problem is the recovery. When I use a similar crispr plasmid (with no guides but a -URA instead of KanMX marker) I get tons of colonies.
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