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Hi Steve,
Firstly, thanks a lot for your prompt reply. I reduced the cycles to 15, and tried either 5min or 7min elongation time. I also spun down the cells and plated everything as you suggested, using high competency bacteria (I always do a transformation control with empty vector to check). Still I get no colonies:( If you have any further tips or ideas for things to change in the reaction conditions, I would really appreciate it.
Thank you.
Hi,
I am trying to clone two 3-4kb sequences into a 7.5kb plasmid and am having some trouble getting the second PCR to work. I get abundant and high purity first PCR product. I am using a KAPA high fidelity PCR mix from Roche with the following cycling conditions:
Initial denaturation: 95°C 3min
Denaturation: 98°C 20s
Annealing: 65°C 15s 25 cycles
Extension: 72°C 12min
Final extension: 72°C 12min
I also tried a couple variations: reducing the annealing temp to 60 + increasing cycles to 35 or decreasing cycles to 15 + 65 annealing temp + 9min elongation time. I either get no colonies or very few which turn out to be empty backbone. I found that increasing the amount of DpnI from 1ul to 1.5ul eliminates empty plasmid colonies and instead I get no colonies at all. Any help would be greatly appreciated.
Project IDs:
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=437b59a76752a5b82ea2b7ac912be788
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=620503d22897aaf36b010296f50ee554
Thanks!
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