Second Restriction Digest of GFP & ATF1 and Gel Check
- Today we did the second restriction digest of our genes, GFP and ATF1. Last time we did a digest using the KPN1 enzyme but could not do the EcoR1 and Pst1 enzymes simultaneously due to a different buffer used for KPN1. So this time, we digested our samples using EcoR1 for GFP to create its "sticky end" prefix and Pst1 for ATF1 to create its "sticky end" suffix. After adding the enzymes, buffer, and water to our samples, we incubated them for 30 minutes at 37°C.
- Finally, we ran an electrophoresis gel to check the success of our digests - how much DNA do we have? The results were fabulous - 4/5 of each of our desired genes, GFP and ATF1, showed very bold and bright bands at the appropriate positions in the gel relative to the 100bp DNA ladder. It was decided that we would ligate them together in a 1:1 ratio.