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Irganomix 11-18-2011 09:24 PM

Restriction digestion small band
 
Hi all
So I have subcloned an 80bp insert into ~3kb vector that is flanked by EcoRV sites. The insert was made by annealing two phosphorylated primers into a blunted vector. I received my sequencing results to verify the insert is correct and now am trying to release this insert from the plasmid to clone into another plasmid.

Since the insert is so small, I digested 10 ug in hopes that the 80bp piece would be strong enough on the gel to remove it--in a 50 uL reaction with 2uL (40 U) of EcoRV for 3 hours and ran it on gel against undigested control. The undigested runs mostly as a supercoil; the digested plasmid has a clear and as expected very strong band around 3kb but nothing below that.

I have previously attempted to PCR the insert from a plasmid but this was somewhat unsuccessful as my negative control without DNA template, i.e. primer dimers only, runs nearly the same region as what may be PCR product but may also just be primer dimers.

Any suggestions?

obama 11-20-2011 12:19 AM

Re: Restriction digestion small band
 
In what percentage of gel are you checked the 80bp release.

And donot keep the digestion mix more than 1hr, since the expected band size is 80bp it may degrade if you keep digestion mix long time.

mmorgan 11-20-2011 07:57 PM

Re: Restriction digestion small band
 
Quote:

Originally Posted by Irganomix (Post 437246)
Hi all
So I have subcloned an 80bp insert into ~3kb vector that is flanked by EcoRV sites. The insert was made by annealing two phosphorylated primers into a blunted vector. I received my sequencing results to verify the insert is correct and now am trying to release this insert from the plasmid to clone into another plasmid.

Since the insert is so small, I digested 10 ug in hopes that the 80bp piece would be strong enough on the gel to remove it--in a 50 uL reaction with 2uL (40 U) of EcoRV for 3 hours and ran it on gel against undigested control. The undigested runs mostly as a supercoil; the digested plasmid has a clear and as expected very strong band around 3kb but nothing below that.

I have previously attempted to PCR the insert from a plasmid but this was somewhat unsuccessful as my negative control without DNA template, i.e. primer dimers only, runs nearly the same region as what may be PCR product but may also just be primer dimers.

Any suggestions?

If you can order your fragment as an oligo, why have you cloned it into a plasmid and then digested it out?

Can't you just order whatever sequence to need and then clone it directly into your second plasmid instead of cloning it into the first vector and then doing another subcloning step?

If the sequence is too long to get as a single oligo you can design 2 fragments and put them into the plasmid together (I can explain how to do this if you're interested). Using 2 synthetic you can generate about 200bp of whatever sequence you like. I expect that if you play around with the technique a bit more you could make even larger synthetic sequences using more than 2 fragments, although it might have to be done is a step-wise manner.


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