Anything and everything cloning: Go...
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Hi Steve,
I have sent you a mail with the details of my secondary PCR condition. I used a 50ul volume for my 2 PCR.
By primer dimer I mean a band near 400bp region. I got 6 colonies in total, I isolated the plasmid and digested with a restriction enzyme to check the plasmid. Out of 6, 4 gave me positive result with restriction digestion, so sent them all for sequencing. I will mail you the details of my protocol and an image of the gel.
Hi Steve,
I have saved my project under my profile. I tried using Phusion with the protocol given on the website. For my primary PCR, I got a nice band, which I excised out and purified. I got about 1ug DNA for the megaprimer. For my secondary PCR, I am getting primer dimers for both 2 step and 3 step PCR protocol. I transformed with the DpnI digested product, but got back parent plasmid only.
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