IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/08/10
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August 10, 2010
I was saddened to see that none of my transformations worked!! I went back and ran a gel of all of my digests to see what was going wrong. Here is the picture:
I found that one of my most important parts; the B0015 terminator, was not showing up on the gel. I decided to use Erin's B0015 for my third attempt at transforming these genes. So I did a lone digestion of her B0015.
Digestion of B0015 (labeled as RXN 0)-
I let the digest incubate in the 37 C bath for 15 minutes, then put it into the80 C bath for 20 minutes.
I then retried yesterdays ligations using the new B0015. All of these reactions are from 8/9/10 <-crazy!
This time I used heat shock to transform cells. I used 50 uL of the heat-shock competent cells and 2 uL of my ligation. I put the mixture in 2.5 mL centrifuge tubes and put o nice for 10 minutes. Then put in 42 C water bath fro 45 seconds, and back on ice for 2 minutes. Then I added 500 uL of SOC media to the tubes and put in the 37 C room for 1 hour to recover. After I took those out they were plated on CAP plates and put into the 37 C room for overnight.