User:Anthony Salvagno/Notebook/Research/2009/02/16/gDNA digestion Try 2

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Last week we had 3ug gDNA in 25uL with 1.5uL XhoI. We found that the enzyme didn't cut as well as we had hoped. (Kelly reasoned that the DNA was cleaner than we anticipated and so we had too much DNA per RE.) Now we are going to try to cut the gDNA again with different amounts of both DNA and XhoI to see what we should use in later tests.

We are mixing less DNA (2ug) in more total solution (30uL) and adding different enzyme amounts (1.5uL and 3.0uL separately).

Preparation

Gel Prep
Tube Number Vol gDNA Vol and Type 10x Buffer Vol BSA 10x Vol XhoI Vol H2O Total Vol
1 9.4uL NE2/3.0uL 3.0uL ---- 14.6uL 30uL
2 9.4uL NE2/3.0uL 3.0uL 1.5uL 13.1uL 30uL
3 9.4uL NE2/3.0uL 3.0uL 3.0uL 11.6uL 30uL

Results

I didn't run this one for long enough in the fluorescing solution, so it came out like this.
I didn't run this one for long enough in the fluorescing solution, so it came out like this.
Kelly let it sit in solution for way longer so it came out like this.  As you can see, the digest went better this time because there was enough restriction enzyme for the DNA.  We got the expected smear of random fragment sizes.
Kelly let it sit in solution for way longer so it came out like this. As you can see, the digest went better this time because there was enough restriction enzyme for the DNA. We got the expected smear of random fragment sizes.
Steve Koch 00:17, 18 February 2009 (EST): Cool!
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