How to improve the agarose gel? - (Feb/20/2008 )
My recent agarose gel seems a little bad. Why is the either side of any lane very high-light?
Would you please take a look and give me some advice to improve the gel?
The concentration is 2%, the Buffer is 1*TBE.
No wonder you have a problem, the gel is running upside down ;-). It looks to me as if you have high molecular weight DNA in many of your samples, which is failing to move into the gel very far. What sizes are the marker lanes? How did you choose 2%? I'd suggest 1% as an initial guess if you don't know the sizes of your fragments. The white band probably is due to large amounts of blue loading dye in your gel. We switched to using orange-g dye in our loading buffer, which runs ahead of the DNA.
the white shadow is because of loading dye- it is nothing to worry about and won't affect your gel. 2% is high- what size do you want to see? There seems to be some junk in your samples that is smearing out- try purifying the nucleic acids by a column kit or phenol chloroform extraction/precipitation etc. Or it could be that your buffer has been reused too many times or is just not the right pH.
Thank you for your advice, Phage434 and lotus.
The lanes I want to see are just the 4th and the 6th from right, with right marker being the 1st lane.
the marker is 50bp, 100, 150, 200, 250, 300, 350, 400, 500 from up to down. The gel is about 2%.
The 4th band is 179bp and the 6th is about 320bp.
They are pcr product.
Should I improve my PCR process to reduce the high molecular weight DNA or improve the agarose gel?
Which is more important, just referring to the 4th and 6th lane?
How large is this gel? It has a strange aspect ratio, and I would guess that you may not be running the gel long enough. Is there a lot more gel with nothing on it at the top which we are not seeing in the picture? It also looks as if you are loading way too much DNA into the well. Try doing some dilutions. If you are looking for a single product band, then the PCR also has problems. Try a higher annealing temperature or redesign the primers.
To me the problem is not the gel picture, but the fact that you don't seem to have PCR product. Try using less DNA template (use a smaller volume or even do a dilution if needed) in your PCR reaction. I would also suggest to pay attention to the exposure when you visualise the gel. Hope this helps a bit.
Thank you, f2dU.
I believe it is worth a try to dilute the cDNA in PCR.
Maybe the gel hadn't polymerised well.
You are right.
It was another problem of mine.
There are several problems:
1. as said above to much template in reaction
2. load less sample in gel
3. The running buffer was too dirty to much runs, change it at least 1X week if low- medium usege if to many people use it during week change it 2X week
4 the white shadow is EtBr that wasn't well distribute in the gel, stir very well or use a shaker (be careful to not use to much rpm or the erlenmeyer will "fly")
One last piece of advice: if you are using EtBr in your gel (rather than staining your gel post-electrophoresis) be careful not to run your gel for long, because small fragments (like your expected products) will get de-stained and you may not be able to see them under UV. This happens because, during electrophoresis, the EtBr migrates towards the negative pole whereas your sample migrates towards the positive. Good luck!