Shivani Gupta-Week 5- Reddy Lab

On Monday, we got back our sequencing of the BirA-YY1 and BirA-L2B which we sent in last Friday. We used software known as BioEdit to align our sequences with the sequences we were supposed to obtain using the original construct we built on Pdraw32, as shown below. 

Unfortunately, when we got back our sequencing from this plasmid, it aligned in the reverse order. We then realized that we chose the wrong enzymes for the double digest as they were not in frame. We then chose two other enzymes to do the double digest with, BamHI and PmeI. Because the BirA-L2B was in frame, we are going to start the maxi preps for them next week. Below is the new construct for BirA-YY1.

We then spent the rest of the week re-doing the cloning. We first had to digest the two plasmids, BirA-Myc/BioID AND PcDNA3-YY1 with enzymes BamHl and Pmel. To do this double digest, I also had to add 10x BSA, NEB buffer 4, and enough water to make each sample 25 ul. After I incubated the samples for an hour, I ran the samples on an agarose gel containing Ethidium Bromide. Because the fragments were the correct size (6382 bp  and 29bp for BirA-Myc plasmid and 1372 and 4751bp for pcDNA6_YY1), I could then run the samples on a SYBR safe gel. I then isolated the fragments by cutting them out of the gel and began the gel extraction protocol. After I isolated the DNA, I ligated the two fragments together using the NEB 5 min ligase. I then did the transformation where I transformed competent DH5 alpha cells with my ligation reaction. After the transformation, I plated them on LB-amp plates. If the colonies grow successfully, we will then do a mini prep and a restriction digest verification/sequencing.