Shivani Gupta-Week 4- Reddy Lab- Cloning Troubleshooting

My PI and my mentor have been on vacation these past two weeks. While it was initially difficult to conduct all the experiments by myself, it became a good opportunity for me to become more independent and comfortable around the lab.

                This week I continued with my cloning project of BirA-YY1 and BirA-L2B. On Monday, I imaged the gel with the BirA-YY1 fusion and the BirA-L2B fusion to verify the restriction digest I did last week. The image of the gel is below. The first six rows are BirA-YY1 and the last 6 are BirA-L2B. 

As you can see, while the BirA-YY1 fusion clearly showed two cut sites, with the correct sized band lengths (1743 and  5895bp), the BirA-L2B fusion showed no cut sites, which meant that either the cloning did not work or the enzyme was not working for Bira-L2B.

 Since the BirA-YY1 fusion was successful, I sent in the YY1 samples for sequencing to verify the cloning. Because the cloning for BirA-L2B did not work, I redid the mini preps for BirA-L2B. To do the mini preps, I first grew up 3ml mini cultures of LB and Ampicillin overnight and added in the bacterial colonies of L2B. The next day, I centrifuged the bacteria colonies in LB and amp. I then removed the supernatant, and resuspended the bacteria pellets in P1, P2, and P3. After I added the isopropanol, I centrifuged the sample again, and removed the supernatant. I then air dried the pellet and resuspended it in EB buffer. To calculate the right amount of DNA to add for the digest, I had to use the nanodrop spectrometer to figure out the concentration of each sample of L2B. After I figured out the concentration, I calculated what the appropriate amount of buffer, DNA, enzyme (we used Scal to verify the BirA-L2B fusion), and water would be to make my samples 1 ug and 10 ml solutions. After the digest, I did the restriction digest verification again by running each sample in the gel. Unfortunately, I had the same result, no cut sites, which concluded the Scal enzyme was not working. Below is the picture of the gel, with no cut sites again. 

 Since the Scal enzyme was not working, I redid the digest with another enzyme, Nhel. Thankfully the Nhel cut the samples at two locations with the appropriate length, proving that the digest was successful.  Here is the picture of the gel with two cut sites, and band lengths of 3237 and 4582bp. 

On Friday, I sent the L2B samples for sequencing to verify the cloning worked. If both the BirA-YY1 and the BirA-L2B come back successfully from sequencing, I will then do a maxi prep to prepare a large amount of the plasmid.

While this week consisted of a lot of troubleshooting, it was important to learn how to re-trace my steps, and come up with different solutions to  resolve what went wrong during the procedure.