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Hi,
another thing: what did you mean by :"you're trying to remove the better part of a KB of parental sequence".
indeed the deletion /replacement that I try to do is about 700bp, is is too long? an and the insert is 672bp long.
it would be graet if you could comment on it.
Hyla
Hi,
thanks for your reply. the 2nd PCR reaction are as the following:
I then set up and ran the secondary PCR with the quantities of insert: plasmid and elongation time indicated in the website using the 3-step protocol. I used Phusion polymerase.
The cycle was as follows:
98°C 30"
98°C 10"
61°C 30''
72 °C 3'
72 °C 5'
72°C 5'
total of 35 cycles
would you suggest me to try 2 step PCR?
Thanks
Hi,
I used the website to design the primers for 1st PCR. You can see the details here.
I sequenced the 1st PCR product and it seems that I have it correct however after the 2nd PCR (tried different annealing temp 61 to 68), I get colonies only with parental plasmid.
I would appreciate your tips and help.
the insert size is 672 bp long.
Best,
Hyla
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