G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/11/10

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Restriction Digest of Amplified Biobrick Parts

  • Today, we began a restriction digest of our amplified Biobrick parts which, through PCR, have now been removed from their vectors using VF and VR primers. The next step was to run a digest cutting each part to allow for later ligation and insertion into a biobrick vector. To run the digest, EcoR1 (prefix), Pst1 (suffix), and KPN1 restriction enzymes all needed to be used. However, KPN1 required a different kind of buffer and therefore had to undergo a separate reaction (which is what we did today). After making a master mix for sixteen reactions (thirteen PCR reactions, one positive control, one negative control, and one extra) containing KPN1, DNA,Buffer KPN1, and water, the samples were incubated at 37 degrees Celsius for thirty minutes. Finally, we performed an ethanol precipitation on the sample to allow for DNA precipitate to form and to use later in restriction digests containing EcoR1 and Pst1 on Tuesday. Friday afternoon we added 20µl water to our samples which had been air drying over night and put our samples in the fridge.