Wednesday, March 28, 2007

Thou shall gel-purify PCR products!

Because otherwise you might just end up like me learning it the rough way. Admittedly, it involved the interplay of several «unfortunate» properties, but nevertheless it eventually made me streak 320 colonies and even worse cost a lot of time. You may wonder what my story is? Here it comes.

80 nicely streaked colonies It all started off with happily glaring PCR products bands implying that 99.9% of the DNA in my solution should be my product. The strategy planned an extra cloning step in very handy TOPO blunt vectors (Invitrogen). This is where my first fault occured: I decided not to gel-purify the PCR reaction due to the high product content. My second fault was to use ampicillin instead of kanamycin or zeocin plates as the manufacturer's instructions would have suggested [1]. Combined with an extremely high transformation efficiency (10 billion per µg DNA) of the Mach1 E.coli cells (Invitrogen) in use, this perfectly selected for the few ng of PCR template. To my defence; I didn't know at the time that my template was a mammalian expression vector carrying genes for ampicillin and neo/kanamycin resistance. A round of weird digest, however positively confirmed inserts by internal primers, and a couple of maxi-preps later I had a huge amount of template DNA, with - in my eyes - still very weird digest patterns [2]. The testing continued, extended to the restriction enzymes and their according buffers, and culminated in a freshly ordered batch of them. The results stayed unaltered though.

Redoing transformations on kanamycin strangely didn't provide any better results. In this case, I think Fortuna refused to guide my colony picking hand and I ended up mini-prepping template plasmids once more. After the flaw eventually became apparent, I played streaking-slave and had a lot of fun with roughly 320 pipette tips, the same amount of E.coli colonies, a grid, and 4 zeocin plates [see picture]. Plasmid DNA purified from the fittest cultures finally led to correct digest patterns. Oh, the joy of trouble shooting.

The morale of the story? Always gel-purify your PCR products! It saves you a lot of troubles. It's better to invest more time and money to do something completely right, than loosing time, opportunity, and money by having to redo something.

[1] This is the reason why one should always read that damn thing, or at least skim it.
[2] As I still believed I had my TOPO vector with the appropriate insert.

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