Shivani Gupta- Week 3- Reddy Lab

In the beginning of the week, I fixed and permibilized fibroblast B63t3 cells (from mice). I incubated these cells with a primary antibody used to mark the nuclear periphery against Lamin B1. To detect this antibody, I used three different secondary antibodies, which are the images below.

 

The last image is a stain called Hoechst (blue one) which stains the DNA in the nucleus.  The darker blue regions are densely packed DNA or heterochromatin, and the lighter blue regions, or where there is no blue, are less tightly packed DNA. 

                After I finished the immunofluorescence, I continued with our cloning project. For the BirA fusion cloning with YY1 and Lap2B, we first had to do a digest with enzymes Kpn1/Xho1 for YY1 and  EcoRV/KpnI for L2B. After the digest, I did a gel isolation to isolate the BirA, L2B, and YY1 vectors.  I then did a ligation which is the final step in building the recombinant DNA. The ligation connects the DNA into the digested vector.  After the ligation, I had to do a transformation which gets the recombinant DNA from the vector solution into E. Coli. After the transformation, we have a plate with bacterial colonies. Here is the picture of the bacterial colonies below.

 

Each of the colonies contains different plasmids. I then isolated the DNA from each colony and analyzed the structure of the plasmids with restriction enzymes. On Friday, I did a mini-prep where we grew up three mini cultures to isolate the plasmid. After the mini-prep, we did a restriction digest verification to verify that the BirA-L2B fusion was made. I did the restriction digest with enzyme Xmal with YY1 and Scal with L2B.  I just ran the gel on Monday to verify the digest and we are planning to image it on Tuesday. Hopefully, I get 2 fragments for both the YY1-BirA fusion and BirA-L2B fusion with the right size, which would prove that the digest was successful!