miniprep DNA v PCR product, for sequencing - (Oct/21/2006 )
I have some clones I need to sequence. I am curious what the pros and cons are for miniprepping the clones and sequencing supercoiled plasmid, versus amplifying my insert from the clones and sequencing that.
Is one method or the other generally more reliable? Is either more rigorous? We typically sequence from PCR products, but are having difficulties at the moment, and if miniprep DNA is more reliable I'd be tempted to go that route.
Would I need to phenol extract or RNAse the minipreps?
In general, preping DNA from a clonal bacterial culture is best. The reason is that PCR products can amplify different sections of DNA (heterologous genes in diploid species, or different rRNA genes in a bacterial species, e.g.) and you will get a mixed sequence in the resulting reaction. A single colony transformed with a plasmid arises ideally from one molecule, and you sequence that particular molecule, with no opportunity for confusion.
PCR products also have the problem that primer-dimers (or anything else incorporating the primer sequence) will confuse the sequencing reaction. A nested sequencing primer can help here.
No special thing needs to be done to minipreps. Any column based prep will work fine, but I would avoid the quick preps with GET/SDS-NaOH/KOAc/EtOH precip that some people use. RNAse is not necessary, but may be important to get the quantification correct. Don't use too much DNA. In general, too little is better than too much DNA in the sequencing reactions.
I *won't* use a kit. I'm old enough to remember when kits first came out, and having cut my teeth on chemical minipreps I've always been of the opinion that kit DNA is never as good as Maniatis protocol # whatever-it-is.
I've used kits in three different labs, for multiple PI's, from different vendors, and bent over backwards to get the DNA as clean as a standard chemical miniprep. They have *never* cut as well as an old fashioned miniprep. They are marginally more expensive. Because of my years of doing minipreps the old-fashioned way I am very quick at them, and so kits are also not faster in my case. They use more plastic.
I only use them when someone I have to show says "Try it you'll see, use a kit" and I have to show them that the DNA is not as good as it is in a chemical prep.
The spin column in the kit always elutes some small bit of flocculent stuff, sometimes it is hard to see but it is always there. An old fashioned prep may have extra salt in it, but no flocculent resin phnewy stuff.
*See foot note
OK, with that rant out of the way (nice to meet you, ) I'm wondering if the RNA might lead to spurious priming or something. It seems problematic to leave so much complementary nucleic acid around. Does it really not matter?
Feedback on another forum suggests that a phenol extraction would be necessary. I don't think that it should be, but I don't know. It seems that residual phenol muight inhibit the DNA polymerase in the sequencing reaction.
I'm also trying to recall the correct amount of DNA to add... is the guideline that you try to sequence about a microgram of plasmid DNA? Or is that way too high?
Thanks for your input.
* The problem I'm concerned about from the PCR is not nonspecific products (we've standardized conditions and our products are usually single - species) but rather whether all the stuff that comes along - Nucleotides, old primer, primer dimer (as you mention), and so on - whether these itty bitty things might screw up the reaction. I could convince myself that miniprep DNA is a better template but I'd feel better having someone with working knowledge tell me if there is a real difference between the two, even if it is only theoretical. your answer goes part way to that. Is it definitely fair to say that a miniprep is more reliable?
Any prep you do will likely be fine. Phenol will inhibit reactions, but it sounds as if you know enough to get rid of it. The sequanase enzyme (basically a pcr enzyme) has little or no reverse transcriptase activity, so even primed RNA won't produce sequence. I suppose some RNA could act as a primer, but I've never seen that be an issue -- the primer is present in overwhelming excess. Normally a 3-5 kb plasmid should have about 150-300 ng of plasmid DNA in the sequencing reaction.
thanks again Phage.
I've been racking my brains trying to recall a method that I used about 15 years ago - I wonder i f anyone here can help.
It was a (basically) standard alkaline lysis miniprep, but there was a cesium chloride step added. There was no high spin/banding involved, the cesium was some sort of step that helped precipitate DNA more differentially than NaAc or other salts.... or something.
I can't find this protocol online, and I am wondering if this variation to the standard miniprep procedure rings any bells to you or anyone else?