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T7 and M13 primers two band amplification


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#1 FJRCC

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Posted 12 September 2013 - 07:00 AM

Dear all,

 

 

I´m trying to check if the cloning of a fragment went well. I´m running a pcr with primers T7 and M13 but instead of a unique band I´m having two bands in my gel.

 

Does anyone had this problem in the past?

 

Any clue?

 

Thanks!



#2 jerryshelly1

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Posted 12 September 2013 - 09:18 AM

Did you fragment have two unique RE sites? Maybe you have a double insert of your fragment.  What are the sizes of the two bands and what is the size of your fragment? How far away is the T7 and M13 from your insert?



#3 FJRCC

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Posted 12 September 2013 - 09:49 AM

Thanks for the answer jerryshelly.

 

The sizes of the bands are approximatelly 700 bp and 300 bp. The size of the fragment is about 400. The 700 bp is surely the fragment (400 of the insert + 199 of the fragment without the insert + the primers). However the 300 bp...

 

FJRCC



#4 phage434

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Posted 12 September 2013 - 10:56 AM

The sequences of these primers are very common on plasmids. Your base plasmid may already have a region flanked by these primers. The T7 primer, in particular, is also the sequence of the T7 promoter. Also, you really need to be more specific (not here, but in your thinking) about the M13 primer. There are at least 4 common versions, forward and reverse, and binding to different locations. It helps to be specific.






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