Week 8 – DNA Fragment Lengths
Hello everyone,
I was unable to access the lab this week because my on-site mentor was traveling. Instead, I worked on understanding the DNA fragments from my vector+insert and created a PCR procedure for when I return to the lab.
First, I looked at the vector (pBABE-neo) to analyze the fragments it would form when cut with PstI (the restriction enzyme used for my gel in the previous post). As shown in Figure 1, I used EcoRI (blue) to insert the gene with a PstI cutting site (indicated with the red on the insert) on it. This makes the distances between each red PstI restriction sites different depending on if the insert is in the correct orientation or not.
As a result, I was able to calculate the number of nucleotides between each PstI restriction site as 3384, ~985, ~900, 872, and 170 from largest to smallest. These correspond to the bands of DNA in the PstI digest gel in my previous blog post. After comparing the DNA bands in the gel with these numbers, I saw confusing mismatches where only some of the bands are in their correct spots.
This week I think I realized cancer research requires a lot of prior knowledge such as understanding how a vector insert like this works. Thank you for reading my blog post and feel free to comment!