9 replies to this topic

[mad]

Hi all,

Hope someone can help me with my weird week.

I tried running DNA thru 1% agarose gel and for some unknown reason they wont run. I tried many times and I keep getting weirder results.
1st time I tried both loading dye and (I assume because I checked with the UV) DNA did not run.
2nd time I tried the loading dye ran but no DNA was seen under UV

Both times were different newly prepared gel, so I am not at a total lost.

MAD

[jiajia1987]

Did you check the composition of the gel? Is it really 1% or did you make it a higher percentage  by mistake?

Did you cover the gel with enough buffer? Try not to overload it with buffer though. What was the voltage and time?

[mad]

both gels were freshly made and I've done it tons of times.
I was testing the gel run rate at 45v for an hour (Don't ask me why my insane supervisor is the one behind this idea), but i've already previously done it before with success.

Oh I forgot to mention that I was running it in a smaller tank from the usual equipment (Soz but the IP crap is making it hard for me to explain my problem)

MAD

[HomeBrew]

I presume these are EtBr stained gels?  I further presume that you're looking at them with the right wavelength UV, and that the power supply was set for constant voltage, and the leads are set correctly (all mistakes I have made over the years)?

Is the power supply working?  Do bubbles form along the negative wire in the chamber?

Is the sample DNA still visible in or near the wells?

Did you include a ladder, and did it run and was it visible?

[mad]

How do you check if the DNA is still in or near the well?

I've looked under the UV and can see a slight brightness at the well. Is that it?

[gebirgsziege]

What kind of DNA are you working with (i.e. plasmid, genomic, PCR-product)?
I agree with HomeBrew: what about your ladder? If the ladder is running correctly, maybe the answer is simple: your PCR did not work?

Usually you can approximate where your DNA is by spotting the dyes...e.g. Bromphenolblue is at app. 300 - 200 bp.

If using EtBr stained gels, you will see the DNA bright only if it ran into the gel. Maybe you should try to add a drop or EtBr to your sample and look at it without a gel in UV light....if there is DNA in it it should be brighter than EtBr without DNA. So you can find out if there is DNA in your sample......

[A_Gisela]

Are you sure your sample is DNA?, could be that you have problems on the isolation method and you are not getting DNA.

Did you check the electrodes?, is your chamber working correctly?

[swanny]

Chuck out your running buffer, make up a fresh solution, prepare a new gel using that buffer and try again.

[mad]

thanks for all the suggestion

I have another question to ask regarding the issue. Does the thickness of the loading dye can have an effect on the sample run?
This is because in this instance I am only getting small amount of product so I cant exactly do a 1:5 (Loading dye:Sample)dilution, so I'm currently doing a 1:1 or 1:2...depending on amount available.

Could this be the problem?


MAD

[warsel]

mad, on Jul 2 2009, 11:11 PM, said:

thanks for all the suggestion

I have another question to ask regarding the issue. Does the thickness of the loading dye can have an effect on the sample run?
This is because in this instance I am only getting small amount of product so I cant exactly do a 1:5 (Loading dye:Sample)dilution, so I'm currently doing a 1:1 or 1:2...depending on amount available.

Could this be the problem?


MAD

possible but not likely. you can also dilute the loading dye to make it 2x instead of 6x of course to counter that problem.

is your maker visible on the gel?
- if it is: 1) did you check to see if your DNA actually stays in the well; in my experience that is usually a problem with samples that have a lot of residual ethanol in them - try to add sucrose to the loading dye to counter the problem.
            2) are you sure there is actually (sufficient) DNA in your tube? if yes - can you exclude that it's just free nucleotides (ie a failed PCR run)?

- if it isn't: check your buffers, agarose, etbr and elpho unit I'd say.