DNA ladder issues- I can't see any :( - (Sep/29/2006 )

Hi

I have recently run a RFLP digests on a 0.5x TBE gel overnight and get bands in the lanes with my samples but nothing at all in the DNA ladder lane. I have repeated the electrophoresis several times with the same result. Others in the lab have had the same problem, but only when running a large gel overnight.I have used the same ladder for a smaller TAE gel when looking at PCR products, it comes up fine.

I load the same amount of ladder to each type of gel (RFLP or for PCR products), but add extra loading buffer to the ladder for the bigger gel. We have considered DNase activity in the loading buffer, however I have used the exact same loading buffer for my samples and bands are visible.

If anyone has any suggestions as to why this may occur, your help would be so gratefully received as I am an honours student in the middle of writing up my thesis and I am extremely worried about how to explain this without sounding like a complete idiot!!!

Thanks in advance

Laura

-lauralee-

I know this is an obvious thing to check for but what is the DNA ladder size range and could it be running off the gel?

QUOTE (lauralee @ Sep 29 2006, 06:52 PM)

-ML1975-

well ...aside from ML1975 suggestion,

I wonder if the concentration of the ladder is high enough to visualise on the gel you are currently using. As it was mentioned no problems were observed with the smaller gel. So I wonder if upon using the larger gel, your ladder bands are so spread out that it is no longer visible.

On another matter, DNAse contamination need not only be in the loading buffer, it can also get into the actual DNA ladder pot, and start working direct there. And DNA ladders are just DNA... and repeated cycles of freeze thawring will given time degrade the DNA (especially for the larger bands)

Always aliquote the DNA ladder.

-perneseblue-

Hi

The ladder is 1Kb+ and is actually an aliquot of a stock that has previously been used for the same type of analysis (RFLP- run o/n) where discrete and fairly tight bands were observed so I'm not sure about whether it could have spread out so far as to be invisible. If this had actually occured though- would you expect to see some sort of smearing??

Also, it is stored at 4 degrees so freeze/thaw degradation is out

I have spoken to my supervisors and several other lab collegues and we are all very puzzled but thanks for your suggestions- I will at least have things to discuss when writing up

-lauralee-

Have you tried to run the gel with the ladder during the day (8 hours) to see what happens? In principle, you could run for 1 hour, take a picture, place the gel back into the buffer, run for another hour and so on for the eight hours. The gel should be fine. I think at the end you should be able to identify the problem.

Other idea is running the gel during less time and higher voltage. If Im not wrong, as a rule of thumb you can double the voltage (being cautious of not overheating your buffer/gel) and it will take half time to run, so in stead of leaving it overnight, you can run during the day and watch it in "real time".

One last thing, people usually avoid writing about the arisen problems during the thesis if they aren't significant, I mean, they aren't publishable. At least your thesis is going really bad because of this and you need to show that you have tried many things during these years, I wouldn't mention anything at all.

Good luck and let us know what you find out.

-gsamsa-

Hi all

Thanks again for all of your suggestions.
Unfortunately it would be impossible to leave this out of my thesis as it was the only RFLP I had time to do. Never mind, I have been assured that it is not what result you get, but your understanding of the process and ability to try and explain what may have happened.
Your suggestions have got me thinking anyways

I'm not actually allowed to do any more lab work but I'll let you know if anyone from the lab manages to figure out whats happening.

Thanks again

Laura

-lauralee-