PCR Gel nonspecific band - (May/22/2011 )
Hi Holly,
You should try my first suggestion first.
Yes, my second suggestion literally means you concentrate the product.
sxh999 on Wed May 25 18:33:36 2011 said:
adrian kohsf on Wed May 25 03:31:46 2011 said:
Hi Holly,
I never do DGGE before, I can't really comment.
The first lane is ladder, second lane very faint, third lane slightly bright with one unspecific band on top.
2 things come into my mind (I suspect): template quality not good, annealing temperature not high enough.
I guess you are boiling your bacteria and use the supernatant as template, am I right? If thats the case, try use a more proper method: CTAB, PCI, Salting out etc...
From your first gel (lower temp), there is no primer dimmer. This your second gel (slightly higher temperature). there are some primer dimmer and one unspecific band on top of your 550bp. This suggest to me your temperature is not high enough, but then I could be wrong.
Since 16S primers usually will work perfectly on 55C, I suspect perhaps your PCR machine temperature had run off. This happened to Biorad Mycycler in one of my labs, when we use temperature as in "algorithmic measurement" rather than "block measurement".
So my suggestion:
1) Do a gradient PCR, from 55 - 64 C to find the best temperature
2) Alternatively, scale up or do a few tubes of PCR, pool together, gel excise your desired band.
2) Do a temperature calibration for your thermalcycler
Hope this helps.
Adrian
Hi Adrian,
Thank you for so much useful information.
My DNA was extracted from the samples using a Kit. The concentration and quality of the DNA had been tested and revealed good quality.
I also tried a new thermal cycler before, but it did not work. so I don;t think it has something to do with the machine.
I will try your first suggestion later.
For your second suggestion, if I understand it correctly, do you mean that just increase the annealing temperature to clear the non-specific bands, and concentrate the product concentration if the 550bp band is faint?
Thank you again and best.
Holly
Adrian K on Wed Jun 1 15:53:39 2011 said:
Hi Holly,
You should try my first suggestion first.
Yes, my second suggestion literally means you concentrate the product.
sxh999 on Wed May 25 18:33:36 2011 said:
adrian kohsf on Wed May 25 03:31:46 2011 said:
Hi Holly,
I never do DGGE before, I can't really comment.
The first lane is ladder, second lane very faint, third lane slightly bright with one unspecific band on top.
2 things come into my mind (I suspect): template quality not good, annealing temperature not high enough.
I guess you are boiling your bacteria and use the supernatant as template, am I right? If thats the case, try use a more proper method: CTAB, PCI, Salting out etc...
From your first gel (lower temp), there is no primer dimmer. This your second gel (slightly higher temperature). there are some primer dimmer and one unspecific band on top of your 550bp. This suggest to me your temperature is not high enough, but then I could be wrong.
Since 16S primers usually will work perfectly on 55C, I suspect perhaps your PCR machine temperature had run off. This happened to Biorad Mycycler in one of my labs, when we use temperature as in "algorithmic measurement" rather than "block measurement".
So my suggestion:
1) Do a gradient PCR, from 55 - 64 C to find the best temperature
2) Alternatively, scale up or do a few tubes of PCR, pool together, gel excise your desired band.
2) Do a temperature calibration for your thermalcycler
Hope this helps.
Adrian
Hi Adrian,
Thank you for so much useful information.
My DNA was extracted from the samples using a Kit. The concentration and quality of the DNA had been tested and revealed good quality.
I also tried a new thermal cycler before, but it did not work. so I don;t think it has something to do with the machine.
I will try your first suggestion later.
For your second suggestion, if I understand it correctly, do you mean that just increase the annealing temperature to clear the non-specific bands, and concentrate the product concentration if the 550bp band is faint?
Thank you again and best.
Holly
Hi Adrian,
Thank you again.
I tried your first suggestion, and it did work once, but it was not reproducible. After many tries, I still could not get satisfied results, so i changed the primers. Hope I can make it this time.
Good luck and thanks.
Holly
HI 
I saw your gel picture, think you should increase the annealing temperature to get more specific band as you desired..
Since you only need to amplify the small fragment as it size ~550bp i dont think it is necessary to repeat the cycle up to 34x
you will probably have better band if just for 30cycles, since your gene just small and reducing cycles can reducing some
unspecific band...try also to optimize the annealing temperature... i usually use 52-58C
I'm concern about your primers since you use to amplify 16srRNA, i did PCR 16sRNA before but hope you know that the best one should be around~1.3-1.4kb instead of ~550bp, so it will give you specific information of your bacteria species
Good luck and you will get the result if you keep trying:)
How do you prepare your DNA sample and how do you store it?