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arsenia 07-28-2008 07:08 AM

DNA does not migrate into the gel
Hi everyone.
I'm quite new to the thing, lousy experience with running gels.
Here's the thing.

I have some genomic DNA isolated from paraffin embedded formalin fixed tissue and I want to see how much it is degraded (formalin fixation really damages the DNA) - how bad the smear will be.

But the problem is, my DNA does not seem to want to enter the gel. It just sits in the wells.

I use 0.8% agarose and SYBRsafe dye (I add it to the agarose before pouring the gel) and 1x TAE, the run goes for 3.5 hrs at 60 V (4 V/cm). The running conditions are what puzzles me the most.

I mix approx 1 uL of loading dye (sucrose, TAE, xylene cianol and bromphenol blue) with 4 uL of the DNA. I cannot tell you the precise amount (varies greatly between samples), but there definetly is DNA in there (concentrations measured with Qubit and all fine) :o

After visualization, there is only some pale fluorescence arount the wells, but no bands/smears. And even my ladder does not show (I'd be glad to attach the picture of this mess, but the attachment manager says that's not a valid image type). Also, the paleness disturbs me a bit, since SYBR is supposed to shine more, as it is much more sensitive than EtBr. But at the moment, that's the minor problem.

Anyway. I first thought it might be something with the buffer, but I see bubbles and the indicator dyes migrate perfectly, just where they are supposed to migrate.

Maybe I'm doing some very obvious error, but this are my first attempts (I did some electrophoresis of PCR products and a RFLP run once during undergrad lab practice). I guess I'm all wrong about the running conditions, but I really don't feel like trying them out over and over and wasting time, agarose and SYBR.

Could someone please help? :spcow:

admin 07-28-2008 08:03 AM

Re: DNA does not migrate into the gel
welcome to the forums :) it has happened to me a long time ago....

it seems to me that either

1) you by accidently made your gel percentage very high (less volume or more agarose? - probably unlikely)

2) you may have very big DNA (unlikely unless its genomic (yup you said it was) and even then it should go into the gel if its 0.8%)

3) you did not run it long enough (usually 30min+ to run small gels - much longer for genomic DNA try to use a smaller percentage gel if possible and run longer)

4) if its genomic DNA you may have a bad preparation with too much unclean DNA (ie membranes, proteins etc - check your ratio of 260/280 in UV spec) that are holding your DNA in the well and not letting it run through.

Try to do this a) run some non-genomic DNA into one lane of your gel b) run positive control genomic DNA (from kit or someone else?) c) take a small amount of your genomic DNA, and either re-purify it or precipitate the DNA again wash it with 70% ethanol, spin down and dry it. Resuspend it in TE or purified H20. Run that and see if it goes through ok.

Can you post / upload your gel. You can attach it in a new post (by Go Advanced).

Cheers :) let us know how you go

arsenia 07-28-2008 08:29 AM

Re: DNA does not migrate into the gel
Thanks Admin.

Actually, I did not worry much about my DNA purity. But maybe I should have.
I used Qiagen DNA mini kit for DNA isolation and then ran some qPCR amplifications. All DNA used (the same that I now want to load on the gel to dobule-check the degradation level) was readily amplifiable, so I figured that it should enter the gel fine (if it's fine for PCR, which is much more a "moody" technique than electrophoresis).

I am used to make a small tick on the flask before microwaving the agarose suspension, so I know how much buffer (well, water, actually) has evaporated during agarose melting and I add the missing volume. So I guess the gel concentration should not be too high.

Sorry, I tried again to attach the pic (uploading from my computer) of my gel, but it still says that it's an invalid file type (7,6 KB; JPEG). Even after resizing. Anyway all you would see is darkness, and a bright cloud all around the wells.

So I guess the only option I have is to retry, but running longer.
After 3.5 hrs, the indicator dyes migrated totally out of the gel and I figured that at least SOME of the DNA should have entered the gel. But it didn't, nor did the ladder.

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