How to improve my nucleic acid gel eletrophoresis?
Need help on my gel electrophoresis.
Main problems I have:
(1) fluffy ends of the expected bands
(2) some bands are too strong, some bands are bit faint
(3) smearing of some bands
(4) How do I eliminate the unexpected/unwanted bands?
The picture attached was running by a 0.8% agarose gel, under 90V, for 50 minutes. The buffer used to make the gel and as electrophoretic buffer was 1x TBE buffer (but it was more than 4 months old - does this matter?)
HOW can I produce a nice gel picture, ideally with consistent bands, nice edges, and not too strong/faint, and NO smearing or distortion or funny shape!!!
- ActionXPotentialLv 41 month ago
making sure you have a consistent amount of DNA will help to standardize across wells. Also, the more you bump up the agarose concentration, the more crisp your bands will be. When I need good quality images Ill bump up the gel to 2% and run for longer. Also, lower voltage will help prevent the frowny face. Although 90 V should be ok (this is largely dependent upon your TBE strength). I have also used old buffer for very long amounts of time with no issues, but it cant hurt to replace it.
As far as unspecific bands, this could be a problem with the PCR reaction itself. Its difficult to know how to fix this without knowing your detailed protocol, but the first place I would start is with the Primers.
1) Unspecific primers = unspecific bands
2) too low of an annealing temperature = unspecific bands
3) too much magnesium = unspecific bands
4) contamination = unspecific bands
5) too many cycles = unspecific bands
6) impure Taq = unspecific bands
7) Bad/degraded Taq = unspecific bands
8) too much primer = unspecific bands
9) having a bad day = unspecific bands
- Anonymous1 month ago
unrelated to personal finance. AZZHOLE.