I have been getting increasingly weak gel electrophoresis bands over the past week following PCR. What might be the culprit? I added my red mix, primers, and H20 in the correct amounts. So is something degrading following multiple freeze-thaw cycles-- e.g. red mix? primers? A truly enigmatic affair!
Thanks in advance!
when you freeze and thaw any solutions you must mix the thawed reagent well or you get concentration problems. The salts freeze last but thaw first so incomplete thawing and mixing leaves an aqueous phase with strong salts and ice with few salts. If you remove the liquid then on re freezing the concentration of everything is lowered so you get good reactions with too strong reagents early on but poor amplification as the salts are depleted.
Primers are also degraded by freeze thaw cycles. I thaw an aliquot and store the primers at 4C for up to a month but the NTPs and enzyme usually need freezing.
If everybody in the lab is noticing this effect then either a bulb in the transilluminator has failed or the filter has failed and you are getting photo bleaching from too much UV light
when you freeze and thaw any solutions you must mix the thawed reagent well or you get concentration problems. The salts freeze last but thaw first so incomplete thawing and mixing leaves an aqueous phase with strong salts and ice with few salts. If you remove the liquid then on re freezing the concentration of everything is lowered so you get good reactions with too strong reagents early on but poor amplification as the salts are depleted.
Primers are also degraded by freeze thaw cycles. I thaw an aliquot and store the primers at 4C for up to a month but the NTPs and enzyme usually need freezing.
If everybody in the lab is noticing this effect then either a bulb in the transilluminator has failed or the filter has failed and you are getting photo bleaching from too much UV light
Thanks Paul; so would you suggest vortexing thawed solutions before working with them?
PCR Redmix will degrade from multiple freeze-thaw cycles. Your primers will eventually, but it will take a lot longer.
How old is your running buffer in your electrophoresis tank? Reusing the same buffer will make your gels have less contrast over time. How does your DNA ladder look? Also, double-check the settings on your camera and transillumunator. Someone might have messed them up. The DNA ladder will give you a good idea if the issue is with the camera.
Dear Katie, thank you for the reply. Ladder was fine and buffer was just refilled 2 days ago. But for some odd reason, I have been getting weak bands whereas other lab members who run on the same gel with me get strong bands. They use a different set of primers and red mix than I do so I am guessing that might be the culprit. We run our PCR's together as well.
Hi ,I would not vortex biological solutions as foaming can remove reagents from solution and it is a bit vicious. For small volumes just flick the tube a few times with a finger and with larher volumes ( more than 400 ul flick and invert the tube a few times. In both cases spin the tube down for a couple of seconds to get the reagents off the tube wall and back to the bottom of the tube
Toss your primers and make a new dilution. Also get a new tube of PCR mix. If that fixes it, then no more worries.
Dear Arsalan Marsani Ahmed, if you have followed all the above recommendations and still no result then you should look through your gel preparation. I mean add EB when the gel solution is cooled down otherwise you will get blur and dispersed bands.
My advcie is preparing small volume aliquotes of any component submitted to freezing and thawing. Besides, this, the DNA sample itself can provide similar problems. In theory, the best option should be thawing each tube just once!. Of course, good mixing is always necessary en each tube, except distilled water ;).
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