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separating mixture of plamids


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7 replies to this topic

#1 vitalgene

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Posted 16 January 2014 - 10:24 AM

Hi

 

is there a way to separate/isolate plamid DNA from a mixture of say 2 or more pool.

 

I think transforming and screening mini preps by sequencing could be a way to go! 

 

any suggestions!

 

Thanks

vg



#2 bob1

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Posted 16 January 2014 - 11:31 AM

That's probably the easiest way.

 

Depending on sizes you could run them through gels and cut out bands.



#3 vitalgene

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Posted 16 January 2014 - 11:33 AM

they are more or less same sizes, resolving wouldn't be good idea!, i will still run them on gel to see anyways!

mean time also setting up a transformation!

thanks



#4 bob1

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Posted 16 January 2014 - 11:46 AM

If you have access to them, long acrylamide gels would be the best for this - the further you can run the gel the better.



#5 vitalgene

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Posted 16 January 2014 - 11:50 AM

we do have the apparatus, do you know what percentage gel? I will try it out by running o/n 



#6 bob1

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Posted 16 January 2014 - 11:54 AM

Depending on size, I would go for about 3-5%



#7 ostiaziocan

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Posted 16 January 2014 - 01:59 PM

Some time ago I separeted two plasmids by selective amplification followed by DPN1 digestion. I agree that this approach does not work in many cases huh.png



#8 rkay447

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Posted 24 January 2014 - 01:35 PM

Find a unique cutter to the plasmid of interest.  Linearize, run on a gel and gel purify linearized (yes, you will still have some of the other plasmids but this helps isolate the one you want).  Ligate, transform and screen colonies by diagnostic digest or sequencing.






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