Shivani Gupta- Week 7-Reddy Lab
Because
our BirA-YY1 cloning didn’t work, I had to redo the procedure. I started with
the original double digest, where I digested BioID (BirA- Myc plasmid) which is
our vector and PcDNA6-YY1with BamHl and Pmel.
I then ran the samples on a .8% agarose gel to verify the digest.
Because both segments were the correct sizes, I then ran the samples on a SYBR
safe gel so I could then isolate the fragments with the appropriate sizes, and
then isolate the DNA from the gel slices. After the gel isolation, I ligated
the two fragments together and then transformed my ligation reaction with
competent DHalpha cells. I then plated them on amp plates and grew tem over
night. About 25 colonies grew on the YY1 plates and none grew on the vector
plates, which was a good sign. Then next day I picked 24 colonies and grew them
on LB and amp overnight and started the mini preps the next day. Unfortunately,
after I did the restriction digest verification, while the samples did cut, the
sizes were way off then what they were supposed to be. The bands were supposed
to be at 1808bp and 5961bp,but as you can see, the bands are closer to 6000 and
8500 bp.
This week, we also continued with
the BirA-L2B fusion. Before we could start the maxi prep, we first had to cut Fugw
plasmid with PacI and EcoRI and then did a plko gfp puro (and NEO) and BirA-L2B
ligation and transformation. Fortunately, lots of colonies grew on the two
vector and insert plates, and none on vector only, so next week, we will then
do the mini preps for them and a diagnostic restriction digest to verify the
cloning was successful.
During
my last week in the lab, we finished the mini preps for the L2B. I mainly spent
the week working on my poster and troubleshooting with Jen, my mentor, as to why
the BirA-YY1 fusion was still unsuccessful. On Friday, my lab held a pizza
lunch for me and gave me a thank you card! Overall, this was a great
experience, and I am so glad I chose to do my EXP at this lab.
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