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I'm looking for a protocol to get genomic DNA from an E. coli sample so that I can clone a small portion of it using PCR into a plasmid. (< 500 bp in this case).

It seems OWW (Open Wet Ware) only discusses preparation of fragmented DNA. Does this mean I have to cut it up before I can clone from it? Any pointers would be really appreciated.

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It's a wiki for describing consensus protocols and organizing lab webpages: – Mac Cowell Dec 16 '11 at 3:25
@shigeta could you elaborate on what you mean by cloning? Do you want to incorporate a fragment of the E. coli genome into a plasmid and then transform the plasmid for cloning? – Mac Cowell Dec 16 '11 at 3:28
Is anyone still looking for an answer to this? Phenol/chloroform extraction is the gold standard. And here's a protocol specific to E. coli genomic DNA that doesn't use phenol/chloroform: – Amy Feb 20 '12 at 21:22
The answer I got from a colleague is that to PCR from a bacterial sample, you can add bacteria right to the PCR tube without even lysing it. maybe this so obvious nobody thought to say this.... – shigeta Feb 22 '12 at 9:12
@shigeta Oh yeah, I do that often when screening colonies following a ligation/transformation. Stick a colony with a toothpick, stir in some water, use the water for PCR. I increase the first 95C step of a PCR protocol from 2 min to 4 min. These reactions aren't the cleanest though - if you amplifying a gene for downstream cloning, I'd still recommend purifying the DNA first. – Amy Feb 22 '12 at 18:30

2 Answers 2

Yes, you must fragment the genome in order to insert it into a vector for cloning; you can't "insert" the whole 5 Mbp genome of E. coli into a vector. It's difficult to transform cells with huge plasmids, 2-20 kbp is an optimal range. In any case, if you want a clone of the whole genome, wait 30 minutes and the cell will happily oblige you.

Most procedures that isolate genomic DNA will fragment it in the first place, as it is much too large and fragile to stay together, and if it did it, the majority would be caught up in other cell debris and discarded. Vortexing with glass beads is a typical first step to randomly fragment it. After this, you can digest the DNA and your vector to place matching ends on it, ligate it, and transform host cells.

If you want a targeted approach (to extract a specific gene, a technique with which I am unfamiliar), you may be able to use PCR on your extracted, fragmented DNA, as there would hopefully be one intact segment spanning what you want.

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To clone a specific gene, you have to design PCR primers that will amplify the gene and also with specific restriction sites flanking your gene. The restriction sites should correspond with the restriction sites found in the poly-linker of your plasmid. For ligation, digest your plasmid, treat with CIP (to reduce vector background), and ligate it with your purified and digested PCR product. Needless to say, this is probably an oversimplified explanation of cloning; let me know if you want more details. – jp89 Dec 20 '11 at 17:08
@JamesPan I've subcloned genes between vectors, I just didn't know if there was any differences between vector->vector and genome->vector. It's all DNA so in theory there isn't anything new, but I don't know about the practical considerations. – Nick T Dec 20 '11 at 17:26
i thought cloning meant to PCR a fragment of the genome - i've never head of popping the genome fragments into a plasmid directly - you would have to have a lot of genomic DNA to do this - sounds hard. – shigeta Dec 20 '11 at 18:00
@NickT I think genome->vector should still be the same general principle. Amplifying genes from genomic DNA should still be straightforward (I amplify genes from human DNA all the time). I find that sometimes, at least in human DNA, it can get fussy at you, so I try different PCR additives like DMSO or Betanine. Use ~50ng of E. coli genomic DNA for your template, amplify, get your products, and insert it into a vector just like anything else. – jp89 Dec 20 '11 at 19:43
@JamesPan feel free to post another answer; your experience and depth of knowledge seems better than mine. The more answers, the better. – Nick T Dec 20 '11 at 20:39
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Hey all I think I didn't know how to word this question properly. After asking a colleague I was told that I don't need to prepare the DNA at all for this tiny non-library scenario.

She told me to just take a little smear of bacteria on a toothpick and put it into the PCR reaction with the master mix and the enzyme and the heating cycles would do the rest.

Maybe this question was too simple - sorry if it was.

( Sure enough it worked fine. )

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