Hello, if possible can you provide information about aprox rage kbp of DNA, % of agarose in gel.

make sure that the gel is fully set after pouring and run it much slower (lower voltage) than you are doing now ,Your results are good and do not need running that far and slower will give better resolution

I think you should provide us more information about your agarose gel electrophoresis. As your agarose gel appeared cloudy after viewed, I suggest that you should heat the agarose&TAE buffer sufficiently to make it fully dissolved. And I want to know which way do you use to stain the gel? Add the EB into the gel solution to heat together or stain the gel after electrophoresis? If the latter, the gel maybe also seem cloudy.

Folllow

Thank you so much for your helpful feedback.

Karen A. Darbinyan Paul Rutland Qi Liu

I'm doing triplex PCR with target gene size 520 bp, 321 bp and 234 bp. For gel electrophoresis it is 1.8% agarose gel in TBE buffer with Sybr Safe stain and run for 45 mins at 100 V.

Hello, for better resolution increase agarose content till 2%, and decrease voltage to 75 V.

Good luck

Try running the gel at 60v for 45 mins but your problem is the pcr not the gel.The smaller 2 amplimers are very weak. You could try lowering the annealing temperature 4C in case the primers are annealing badly and adding slightly more magnesium ( half as much more than you are using now) in case your dna has pcr inhibitors present. If that fails try with more of the small product primers in case the primers are becoming degraded. Your pcr is basically good with no primer dimer so you can make pcr conditions more relaxed without worrying too much about non specific amplification. When you have bands for all amplimers you can make the pcr more stringent to clean it up if you need to

do you mean to say LB Ruslan Kalendar ?

1. Initially you run your gel on low voltage

2. Before removing comb, you can keep gel in buffer for 5 min

3. Try to load ladder at second or third well... see if you get any difference ...