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Why do I get two bands on the gel of a digested PCR product after purification w

molecular cloning digest pcr purification

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#1 Delimar Rodriguez

Delimar Rodriguez

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Posted 27 April 2018 - 01:05 PM

Hello friends.
 
I am trying to clone a region of 80 amino acids with a weight of 265 bp in pET28a.
When I perform the PCR of my insertion, I see a single band.
Perform the digestion of the insert with the NheI and BamHI enzymes and, when you check the result in agarose electrophoresis, show a single band corresponding to 265 bp.
 
Then purify the digested insert of the agarose gel using a kit. Take 5 ul of that purification and look at it on an agarose gel to verify the purification. Now two very close bands appear instead of just one.
 
What do you think may be happening?
 
Is it advisable to use this product to link it to the vector?
 
Thank you in advance for your help.

Edited by Delimar Rodriguez, 27 April 2018 - 04:22 PM.






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