Anonymous
Anonymous asked in Science & MathematicsBiology · 1 month ago

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!!!

Please help

Attachment image

2 Answers

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  • 1 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

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  • Anonymous
    1 month ago

    unrelated to personal finance. AZZHOLE.

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