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#1 2017-12-18 23:08:54

abhinit
Member
Registered: 2017-12-18
Posts: 3

Secondary PCR trouble

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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#2 2017-12-18 23:43:22

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 110

Re: Secondary PCR trouble

Hi Ahbinit,
Okay, I see your project. The plasmid is somewhat large, which makes it a bit more difficult. The primers themselves look fine to me though (better than most, actually). How many colonies did you see after transformation, and how many did you send for sequencing?
Also, when you say primer dimer, what exactly do you mean? A smear around ~700bps? It might help if you send me a picture of your gel.
It will also help if you write out exactly what all of your methods were. It's difficult for me to help troubleshoot without that information (i.e., all PCR reagent volumes and exact cycle conditions).
-Steve

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#3 2017-12-18 23:52:43

abhinit
Member
Registered: 2017-12-18
Posts: 3

Re: Secondary PCR trouble

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.

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#4 2017-12-19 15:30:38

abhinit
Member
Registered: 2017-12-18
Posts: 3

Re: Secondary PCR trouble

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.

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