BlueGene: The Re-PCRing (and Re-Digesting)

pcr gel ii crop

Following this weekends failed restriction enzyme digest, I thought I would try again with  no heated lid, and with a longer incubation time.  I am using HindIII-HF, so I can apparently let it sit overnight without too much star activity (non-specific cutting).  Since I used quite a bit of my PCR product last time around, I thought it would be a good idea to re-PCR my gblock.  After a picture-perfect gel (above)


Speaking of pictures, here is how I do my gel documentation.  I just shoot my Canon SL1 through an orange piece of acrylic, at the gel.  I set stuff up roughly like this, then I turn off all the lights and take a long exposure.  With this method, I have been able to visualize 100ng samples of DNA regularly, although you can also see bands of half that mass.  I am wary of claiming I can visualize a band below 100ng since it was very faint and I had the aid of another identical lane later in in the gel when I did it, so it was easy to pick out against the background, which let me adjust the exposure to get a better picture of the band.

Anyway, I just put in my digest to run overnight at 37C.  I will heat-inactivate it at 80C for 20 minutes when I get back to the lab, and run it on another gel!  Time to go home!

Posted in: ENG

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