Posted by: ericmjl | July 8, 2009

ssDNA – 3 weeks facing the scourge

So for the past 3 weeks, I have been trying to clone a 100-ish b.p. fragment of DNA into our ampicillin construction plasmids. For 3 weeks, I have faced endless frustration in the face of failure. Today’s revelation about the ultramers that we ordered from IDT was simply a relief off my back.

It turns out that the ultramers are ssDNA strands, not dsDNA strands, so naturally they weren’t cutting well. (I’m half crying and half laughing at my own stupidity as I write this.) That’s why for the longest time I was getting few transformants (1 or 2 colonies a plate), and even those transformants carried a plasmid with little sequence homology to the original inserts. No wonder things were harrowingly frustrating. No wonder Amelia also failed to clone the lock/key/control lock in (I was expecting her to succeed where I had failed).

Now that I know, I guess I needn’t be depressed about my inability to clone that 100-ish b.p. sequence into an Amp construction plasmid. I guess I should move on – after all, my digests certainly look good, and my ability to ligate seems fine.

Onward I go, moving onto the next step – trying to do an actual assembly of new stuff, really exciting stuff.

Update: To rectify this problem, we’ll PCR up the ssDNA using the fresh new primers that we’ve got. That should solve the problem. A sequence check should help us make sure that the parts are fine.


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