Hi everybody,

I am currently working on my Bachelor Thesis, for which I am performing EMSA on CRISPR/Cas9. I am trying to bind Cas to a specific target, but every time I try I get these multiple shifts. I asked around in my department but no one seems to know what these shift can be. Does anyone has an idea of what this is?

Some details about the EMSA:

I target 20 nM of Cy5-labled DNA, with 200 nM of Cas and sgRNA. First I incubate the Cas with sgRNA for 30 min at 25 degrees Celsius, after this I add the DNA and incubate for another 30 min at 37 degrees. I load the 10 uL (5uL 30% glycerol with 5ul sample) on a 10% TBE gel. I rinse the wells before loading and run for 90 min at 100 V.

I run two different target DNA's and each target DNA with two different sgRNA's. On the gel you can see the following:

1) Only target DNA 1

2) Target DNA 1 with Cas and RNA 1

3) Target DNA 1 with Cas and RNA 2

4) Only target DNA 2

5) Target DNA 2 with Cas and RNA 3

6) Target DNA 2 with Cas and RNA 4

I really hope you can help me with my problem!

Menno