DNA virus purification from agarose failed! - (May/09/2014 )
Hello! well I'm trying to purificate a band from a plasmid digestion, the problem:when I try to purify the band of the insert (the virus) I always get a very low concentration (less of 0.5 ng per microliter), i want to re-ligate it to digest it and clone with a diferent restriction site, the other idea... is to "digest to the hell" the actual plasmid and religate the virus, any suggestions?
the virus is a geminivirus...
Agarose extractions are often problematic - you typically need a lot of DNA to get any decent yield, and typically yields are less than 5% of the input amount.
You have the whole viral genome in a plasmid? or is t just one gene?
Either way, it would probably be quicker to PCR amplify the insert with primers containing the restriction sites you want, digest that and then ligate into your new plasmid,
Well, is the complete genome, I want to make a dimer or hemidimer, and the PCR idea is not possible at the moment, do you think, if i digest the plasmid (just the plasmid) can I re-ligate and the cut it again to clone it in another plasmid?
Sure, it is possible to cut and then religate - but I don't see the point - that would just give you the same as your original plasmid with one extra step that would have essentially done nothing. PCR for a 3000 bp insert is relatively simple to do, and definitely feasible.
One way to change the restriction site is to use adaptors - these are short oligonucleotides that can be ligated into your vector or onto your insert so that you have the new restriction site present when you clone. You can get these synthesized (complementary pair needed, make sure you get HPLC or PAGE purified ones) by anybody that makes primers. If you do it correctly you can even make them so that they already have the right ends with no digestion required, just anneal and then ligate.
As your genome is in a plasmid - it is easy to make lots more DNA - all you have to do is some midipreps or maxipreps, which should give you greater than 50 ug of DNA to play with - so you should be able to digest a large amount and load a lot onto a gel (you could do a DNA precipitation to concentrate if needed to get a smaller volume), this would give you more in the band you are trying to purify.
Digest all the plasmid (all the restriction sites that the viral genome doesn't have). When I said re-ligate, I mean to re-ligate the genome -without the plasmid- to cut it again to make a new construction -because the site where it is cloned right now, is useless for a "hemidimer"-, and yep, I'm going to make a maxiprep, maybe on monday :)
Ah, OK, that could work - you would still need to gel extract though, and there are lots of REs that have incompatible buffers, so you would probably need to clean up (just a PCR cleanup) between those that are compatible and those that aren't.
That's great, I will try it!