General Question

ScottyMcGeester's avatar

How can I make this restriction enzyme digest better?

Asked by ScottyMcGeester (1897points) November 14th, 2014

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.

Observing members: 0 Composing members: 0

1 Answer

Here2_4's avatar

I haven’t the foggiest idea what you speak of there, but I do know that staring at a problem too long alters your vision of the problem as a whole. Start your down time by completely disassociating yourself from the problem entirely. Do something completely uncharacteristic for you. Make it something which can fully absorb your attention, and something way out. Car surf, or go shopping dressed like little Bo Peep, or travel to a community you don’t frequent and find a place to participate in karaoke. Spend an afternoon volunteering to be entertainment at a daycare.
I am guessing slides are cleaned well between uses. Do that with your focus. Cleanse it, and come in fresh.
That is the very best I can offer you.

Answer this question




to answer.

This question is in the General Section. Responses must be helpful and on-topic.

Your answer will be saved while you login or join.

Have a question? Ask Fluther!

What do you know more about?
Knowledge Networking @ Fluther