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  2. → Viewing Profile: Posts: HBImolbiol

HBImolbiol

Member Since 15 Mar 2011
Offline Last Active Feb 12 2013 08:06 AM
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Posts I've Made

In Topic: High frequency of vector religation

12 February 2013 - 07:57 AM

View Postphage434, on 12 February 2013 - 07:43 AM, said:

I just want to check that you are cleaning up the PCR reaction prior to cutting with the enzyme. If you don't do this, then the PCR  enzyme +dNTPs will extend the DNA and trash the cut end of your insert.

Yes, I PCR amplified the 6 kb insert from another plasmid and gel purified it with a spin column prior to performing the restriction digestion.

In Topic: High frequency of vector religation

12 February 2013 - 06:34 AM

View Postjerryshelly1, on 11 February 2013 - 06:55 PM, said:

Self-ligation is not favorable, but it can occur depending on the overhang your RE produces and the efficiency of your enzyme.  The good thing is all (pretty sure) neb HF enzymes are used in NEB Buffer #4, which (i believe) is the most suitable for CIAP.  Double check all that.  Just do your digestion (make sure KpnI-HF does not like to remain on ends, if so heat inactivate) and add your CIAP.  Again, I would produce novel cloning sites though.  The above is alot of work.

I think you're right.  I may try one more time but use Acc65I, which cuts at the same sequence, can be heat inactivated (unlike Kpn) but leaves a 5' overhang which should dephosphorylate more efficiently.  In the meantime I'll see if I can't find a way to make this a 2 enzyme subcloning and save myself some grief.

In Topic: High frequency of vector religation

12 February 2013 - 06:27 AM

View Postbob1, on 11 February 2013 - 07:06 PM, said:

A few basic things:

Are you absolutely sure that the Kpn1 is working properly?

If so, check and re-check that the ends you have on the insert primers are the correct ones (primer design mistakes are very easy to make).  It is best to have at least 6 bp outside the cut site

Also try some different ratios of insert: backbone, the more insert there is, the more likely it will be inserted.

Yes I double checked that.  The enzyme site on the primers are correct and I have 6 bp outside the cut site.

View Postalmost a doctor, on 12 February 2013 - 05:32 AM, said:

This might be a silly question but when you say "followed by PCR column purification", do you run a gel and cut the digested vector, or just apply your restriction digestion mix to the PCR clean up kit?  

Also agree with Bob1, do you know for sure your enzyme works?

I am applying it directly to the PCR column (following dilution with the kit's binding buffer) because there is no fragment released with a single enzyme cut.  I find that gel purification in general lowers ligation efficiency because of the UV light damage to the DNA.  I only do this if I have to purify the vector away from a released fragment.  I do run 2 ul on a gel after purification to ensure it is fully linearized as well as for quantification purposes.

In Topic: High frequency of vector religation

11 February 2013 - 06:11 PM

Thanks for the advice... I hadn't thought the vector could religate during cleanup without ligase?

Anyhow...  The vector is 9 kb and the insert is 6 kb.  There are very few restriction enzymes left that don't cut either one or the other but I'll definitely re-examine my strategy to see if I can't somehow make this a directional subclone and re-do the PCR with different restriction enzyme sites.

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