I'm attempting to determine the sex of African penguins (both parents and chicks). I extracted the DNA from feather samples (Chelex protocol) and now using the primer pair 2550F and 2718R (Fridolfsson). I used a the same PCR protocol (Fridolfsson) but with a gradient to see which annealing temperature is the best (50-60 C). Attached the photo of my gel (1.5% TAE, 75 Volt, 30 min). On the top 8 gradient wells is DNA from a male adult placed and top right 8 wells from a female adult (bottom are unknown chicks).
It seems that the annealing temperature does not make a difference, does anyone has a suggestion how to improve the bands on the gel? Furthermore, the first band (600 bp) is more intense than the second band (450 bp). Females should show two bands (450 + 600) whereas male should show only one band (600 bp). On this gel I see several bands (also in some samples of the male).
Does someone have a suggestion to improve this analysis and/or the reason why the second band is less intense?
Hi Anouk,
1. Firstly you have to decrease the primer concentration and annealing time.
2. After this, if the results will not be up to the mark, then you have to use PCR enhancer (Glycerol, DMSO etc).
3. Add DNA marker in the side well, So that you can check the exact size of the amplicons.
PCR amplification is varies lab to lab and chemicals to chemicals and thermal cycler.
good luck
Hi ! Here are a few ideas:
1) organize your samples as follow : 1 adult male ; 1 adult female ; 1 offspring ; 1 adult male ; 1 adult female ; 1 offspring ; ...
In this way each offspring will be surrounded by one male and one female which should facilitate the comparison.
2) Increase the migration time to to increase the band separation
3) Wait until there are adult
4) Test if their are more interested by a car toy or a doll
5) Find some more reliable markers.
Good luck
Hi both!
Thank you for your help (and the funny ideas :P). I have tried today with less primer concentration, higher agarose concentration in the gel and longer running time and the bands are shown as two good bands!
Hugo, I will let you know if the chicks are more interested by the car of the doll :).
Did you standardize your DNA concentrations before PCR? If you have too high a concentration of DNA in your tube, the packed conditions might cause "false priming," in which the primer anneals to a similar sequence but not the right one (False Positive). Using too much DNA in each tube can even disrupt DNA synthesis during PCR because of obstructed diffusion of large TAQ molecules.
Dear Anouk,
I see, that your problem have been solved yet, but I have an idea for the future:
If you has a vertical gel system for polyarcylamide gel electrophoresis, you can use it for nucleic acid separation as well. The resolution is better in most of the cases, and you can prepare gradient gels as well the improve the separation.
Good luck for the future!
Thank you Matthew and Gizella :).
I hadn't the possibility to measure the DNA concentration due to labconditions, but it worked out in the end. In the futer I will try the vertical gel system you suggested Gizella, thank you.
Hi Ralph,
Thanks for your message! I am responding very late as I do not use this regularly. Please could you contact Gwen Traisnel at the NMMU as she is trying to sex African penguins chicks for her PhD. You might know her :).
Cheers,
Anouk
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