Hi everyone, can anyone tell me why my DNA ladders smeared so badly when I ran these gels but the samples are fine?
Also, from what I understand, for both of my gels (I ran the same two primer sets after doing a melting temperature gradient for each but on two different samples; one per image) my 18S primer seems to have bound well (although I will need to run again with a reliable ladder). The other primer I used did not show up for one sample but showed up faintly for the other. The difference in the samples is that the cDNA concentration of the second one was more dilute than the first. Is it possible that primer may work if I try this again?
Hi jaelin.
Frequently reasons for DNA lader smearing:
-Old ladder will start to degrade and result in a smear, try a new one.
-adjust voltage. Maybe it is to high.
Regarding the gradients, could you please indicate the temperatures? As far i see, the temperature range seems to not be the best to identify the best anneling temp
Miguel Fernández-Niño
I have been told by some other people that I asked that it may be because I overloaded the well. I used really small wells on this gel and used 2 uL to load them. I have used this same ladder before, as have my lab mates, on bigger gels and it has run fine. Therefore, I don't think it has degraded.
I am going to try running some ladders on a gel with bigger wells and see if they turn out better. I may also try decreasing the loading volume to see if that helps.
I believe the annealing temps I tested in that gradient were between 55 and 70 degrees, but I'll have to look to confirm that for sure.
What is the configuration of ladder (sizes) and percentage of gel used?
Ladder smearing is because of (1) Degradation of DNA (2) Lower concentration (3) Use of unsuitable agarose gel concentration. Based on ladder sizes the gel to be used should be decided.
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