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Help! Plasmid resistant to restriction digest after acquiring Flag tag

Restriction digest cloning plasmid Flag tag

Best Answer phage434, 13 September 2013 - 08:29 AM

A common problem is reaction inhibitors in your DNA. This is often exacerbated by running reactions in low volume, with the majority of the liquid coming from a DNA prep. Try diluting your reaction with water (adjusting the buffer, of course). Common inhibitors include ethanol, phenol, Gu-HCl.

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#1 KristinaZ

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Posted 13 September 2013 - 08:02 AM

Hello. I have a strange problem. I inserted a Flag sequence into pMX retroviral vector. Now this new pMX-Flag vector has a hard time to digest (only about 20% gets digested) with any restriction enzyme in the polylinker while the old pMX cuts beautifuly. I sequenced the new pMX-Flag clones and there is no problem. I have never heard about such a problem. I am using qiagene miniprep plasmids - can they be dirty and so not cut? Original pMX is a maxiprep.

 

Thank you for suggestion since I am clueless.

 

KristinaAttached File  gel.pdf   671.21KB   82 downloads



#2 phage434

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Posted 13 September 2013 - 08:29 AM   Best Answer

A common problem is reaction inhibitors in your DNA. This is often exacerbated by running reactions in low volume, with the majority of the liquid coming from a DNA prep. Try diluting your reaction with water (adjusting the buffer, of course). Common inhibitors include ethanol, phenol, Gu-HCl.



#3 KristinaZ

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Posted 13 September 2013 - 09:43 PM

Thank you for reply. My plasmid was isolated by quigen miniprep columns and the concentration was 550 ng/ul. I only cut 200 ng so the volume from the original miniprep was very low in the digest.

It seems to me that this new pMX*Flag plasmid likes a certain supercoil sturcture (see on the picture that I attached) and maybe does not let the enzyme to get to the restriction site? It sounds like nonsence but I the mobility of the circular pMX and pMX*Flag is very different?

 

I will try to digest maxiprep pMX*Flag that I made yesterday.

 

Thank you

 

Kristina



#4 KristinaZ

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Posted 25 September 2013 - 04:56 AM

I purified the plasmid from a maxiprep and it digested beautifully. Thanks-probelm solved!







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