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Column for removing short DNA digestion products

Digestion Cloning

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6 replies to this topic

#1 GalaxyEyes

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Posted 03 September 2013 - 11:49 AM

Hi all, I'm currently doing a library preparation experiment in which I have to digest an overlapped PCR product, then phenol chloroform extract to remove the restriction enzyme, then gel extract to separate the short end products from the insert (about 40 bp each). As you can imagine, a Ph-Cl extraction followed by gel purification really kills yield. Is there a column or a kit that will allow me to remove the restriction enzyme AND remove the short end products after digestion? I remember seeing some PCR cleanup kit htat would do this, but I can't seem to find it.



#2 hobglobin

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Posted 03 September 2013 - 11:55 AM

Qiagen's PCR purification kit is doing such jobs, but I guess all companies have similar (and perhaps cheaper) in their product list.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#3 phage434

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Posted 03 September 2013 - 03:49 PM

Heat kill your enzymes if you can (choose enzymes that can be heat killed if possible). I find Ampure magnetic  bead cleanup far easier and quicker than columns for almost all applications. You can set the size of retained fragments by choosing the amount of bead mix you add, but 40 bp is below most cutoffs. Beads work extremely well for large numbers of samples, but I now use them for almost everything.



#4 GalaxyEyes

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Posted 04 September 2013 - 06:43 AM

Unfortunately the enzyme we use can't be heat killed. This is actually something I've looked into; do Ampure beads remove restriction enzymes as well as short products? I've only seen them used with PCR products.



#5 phage434

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Posted 04 September 2013 - 10:49 AM

I don't actually know, since I work only with heat killed enzymes. It's an easy experiment.



#6 hobglobin

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Posted 04 September 2013 - 10:56 AM

For such RE's it is suggested to inactivate them 20 min @ 80°C . Never tried this but may worth a try.


One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#7 student47

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Posted 19 September 2013 - 07:58 AM

not an expert, but i think most kits are designed to get rid of these things (not sure about the short end products ),  cheack kits - zymoresearch, qiagen .. 







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