Anything and everything cloning: Go...
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Hi Steve,
It worked beatifully!
Thank you very much for the advice.
Sincerely,
Tamjeed
Hello Steve,
Thank you for your reply.
The plasmid I am using is a pet42a backbone with my 255 a .a. gene already inserted; so total approx 8kb. The gene was inserted between the Nco1 site and the Ase1 site using RF cloning. (YAH!! that time it worked beautifully!)
The PCR mix was 80ng of plasmid, 300ng of primer,
PCR cycle 95C 2min
Then 18 cycles of
95C for 30 seconds
68C for 9 minutes
then a final elongation stem of 68C for 12 minutes.
I am using Pfu ultra. I use 1.5ul DMSO in the mix. the reaction volume is 50ul.
I don't use the full backbone because its just convenient to enter sequence I get from sequencing the constructs. I have had success subcloning larger fragments before. In this case I want to insert a series of Prolines; maybe that is giving me issues.
I have used RF-cloning successfully several times.
Recently I have attempted to substitute 8 aa using the technique. I have made two sets of primers that are slightly different. Both sets have not worked. I have also eliminated the 55C annealing temperature and gone on with the 2 step protocol (68C annealing and elongation).
My two project files are below
http://www.rf-cloning.org/rf_cloning_pr … becb9fc7a4
http://www.rf-cloning.org/rf_cloning_pr … c1a0bcb569
Is this because of poor primer design?
Thank you
Sincerely,
Tamjeed Saleh
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