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Restriction digestion troubleshooting - (Sep/19/2009 )

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Warren on Sep 20 2009, 08:56 AM said:

biobio on Sep 20 2009, 07:37 AM said:

Warren on Sep 19 2009, 06:33 PM said:

All plasmid preps have a boatload of RNA if you do not get rid of it (digest it). It's not unique to Qiagen. Usually these kits (haven't used that particular one) come with RNase that is added to Solution I or at some point. I wonder if the RNA might interfere with binding on the column though, which could reduce the plasmid yield. I'd check -- I bet there is supposed to be an RNase step early on in the prep that may have been left out (intentionally or unintentionally).

Dam methylation doesn't effect XhoI, so growing in a dam- strain won't help anything. There is a type of methylation that can effect XhoI (I forget what it is) but it is very context specific and usually the DNA around the XhoI site doesn't have the proper sequence. I wouldn't worry about it, XhoI is a good enzyme.

But I do not think there is a problem transforming a methylated plasmid into a dam- strain (someone can correct me if I am wrong). Just not necessary here. Methylation problems don't come into play very often with regular plasmid cloning using common enzymes. Warren..

Yes.. there is RNase normally added to the lysis buffer - it could have gone off or something.. Will find out today or tomorrow.

Thanks for clearing up the specifics about XhoI methylation, I didn't quite get it before.

Another question that comes to mind: I amplified my insert using conventional Taq. I thought that because the gene is relatively small (~1.5kb), Taq would be enough. Do you agree or would you go for a high fidelity polymerase instead? (in which case, could I use the same thermal protocol for amplification?). I understand sequencing of the insert is of limited diagnostic use..

To me its a no-brainer if you are cloning -- use a high fidelity polymerase, the higher the better. 1.5 kb doesn't sound like much but it is a huge piece of DNA for taq to amplify with no mutations, and screening clones by sequencing is a real pain. As for using the same cycling parameters, it depends on your polymerase -- if it was a simple PCR that you didn't have to tweak for taq to work, and you have "good" primers and template, there is a good chance the same parameters will work -- once again it depends on your polymerase though. Warren..

Just to give the update..

I did the RNAse treatment on the plasmid and I saw a small reduction in the bottom band.. So it seems you were right, it was RNA..

In the meantime I went one step back and worked with an old mini-prep of the same plasmid. Cut it again and it all worked as it should.. Let's see I get colonies tomorrow!

Thanks a lot for your help! :wacko:

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