How can I make this restriction enzyme digest better?
I generally test this separation device thingamabobber which I’m not at liberty to say much of but long story short: I take E. coli pellets and make sure I can successfully view the DNA bands in an electrophoresis gel. One of the samples is a set of bands from a restriction enzyme digest called PstI.
The problem is – the digestion bands are SO WEAK. I keep needing to tweak the contrast of the gel image in order to view them. I look at results from my predecessors and most of them are fully fleshed out. However, they wrote very little to explain exactly what they did.
My suspicion is how the restriction digest is carried out, but I don’t know what. These are the steps it takes in a thermal cycler program set specifically for this test:
Step 1 – 37 degrees C for 22 minutes
Step 2 – 68 degrees C for 20 minutes
Step 3 – 4 degrees C for 15 minutes.
And that’s it. Then I’m supposed to take it out and pipette it into a gel.
Could it also be that there’s not enough PstI enzyme I’m putting in? The stock solution formula consists of:
(N= number of reactions)
1 x (N+1) uL of PstI enzyme
1 x (N+1) uL of NE Buffer, 10x
0.1 x (N+1) uL BSA, 100x
2.9 x (N+1) uL reagent grade water
You take 5uL of that and mix it with 5uL of the E.coli DNA in solution, then place it in the thermal cycler.
The tiny amount of BSA was something I always questioned, but nobody here in the lab has really helped me out. They don’t work with this stuff so they don’t know. I have several days of down time so I could play around with changing any of these settings, but I want to have a direction so I’m not totally lost.
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