October 28th-November 1st

Week 6 (Oct 28th-Nov 1st)

On the 28th, we single digested pgK with xbar, inoculated our TGY and LB plates. We also gram stained one of our sonora plates, which looked okay. We decided to throw away our previous transformation tubes since there was no growth.


We ended up ligating our pgRNa and Kana on the 30th using a 5:1 ratio. Typically, we used a 3:1 ratio, but increasing the insert will give it a better chance of ligating since we’ve been having issues in the past. 



Quick T4 DNA Ligase: 1 ul

2X Quick Ligase Buffer: 10 ul 

Vector DNA: 2 ul (pgRNA)

Insert DNA: 3.5 ul (Kana)

PCR Water: 3.5 ul

———

Total of 20 ul


On the 31st, we ran a gel on our previously ligated pgRNA and Kana. If our ligation has not worked, we will then proceed with using lambda DNA as a control group to be able to understand what is going wrong: digestion, ligation, the primers, or even the DNA itself.


For our ligation gel, we used 10 ul of pgK and 2ul of dye.


The pgK on the gel showed us that the ligation did not happen. As stated before, we’ll move on from this to figure out where it went wrong.

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