Last night I designed and ordered the two complex primers I'll need to amplify and insert the SpcR gene. They won't get here until Monday, so today I did column and agarose-gel cleanups on the two other PCR fragments I'll be using
First I pooled what was left of the two PCR reactions (A and B). The volume of each was about equal to the amount I had run in my gel yesterday (left panel below). Then I did a column cleanup to get rid fo the bulk of the PPCR primers, using an old EconoSpin column that I'd revived by passing 0.2 N NaOH through it.
Then I ran all of the eluate from that column in one lane of a 1.2% agarose gel. I used only about 1/5 of the usual concentration of Ethidium Bromide, and I viewed the gel only with our hand-held long-wave UV lamp to avoid UV damage to the DNA. The bands were faint but clear and I cut them out together in one gel slice.
Then I used our new Zymoclean gel-recovery kit to dissolve the agarose and recover the DNA, and ran 3 µl of the resulting 24 µl in unused lanes of the same gel I used yesterday, to see how much DNA I had recovered.
The results look great. The bands are sharp and bright and the right sizes, and the intensities suggest that I've recovered most and perhaps all of the DNA I began with. So now this prep is in the fridge, waiting for the other piece of the construct.
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