PCR Gel nonspecific band - (May/22/2011 )

Hi there,

Attached please find my PCR gel, the marker is 1kb DNA ladder.

what are the problems of this PCR? Non-specific bands or smear? How could I overcome these?

Thanks for any suggestions.


PS: Genomic DNA, DNA template is around 60ng.
PCR reaction system is 25 uL,

Your image cannot be viewed. Even if we could see it, you have provided far too little detail to make any meaningful comments. Tell us everything about the primers, enzymes, cycle conditions, DNA template, what you are expecting.

I honestly don't understand how people expect us to know these things without giving any hint.

phage434 on Mon May 23 00:01:26 2011 said:

Thanks for reply. Sorry for missing information.

Here you go:

25uL PCR reaction system: ( to amplify a ~550 bps fragment of 16s rRNA Gene from each DNA sample)

5*PCR buffer, 5uL
MgcL2(25mM), 1.5uL
dNTPs (10mM), 0.5uL
Primer: 357f (25uM), 0.25uL
907r (25uM), 0.25uL
Taq (5U/uL), 0.125uL
DNA template, ~60ng

The program is:

94 degree, 3min
35 cycles of :94 degree, 45s; 57 degree, 45s; 72 degree, 90s;
72 degree, 7min

For the picture uploaded, I am confused that why you can not see it and I don't know how to attach it, I send the picture to you as a private message, please have a look and give me some suggestion, I will very appreciate if you can post it for me. thanks a lot.

I still cant see your picture. try upload to http://imageshack.us/


btw, your cycling condition looks similar with mine. You can try 27f, 1492r primers as alternative, I have yet to failed with this 2 primers yet.

adrian kohsf on Tue May 24 01:46:06 2011 said:

Thank you so much for your suggestion,

Here is the link of the picture, hope it works.

http://imageshack.us/content_round.php?page=done&l=img204/7104/pcrgel.png&via=mupload&newlp=1

sxh999 on Sun May 22 23:29:59 2011 said:

Here is the link of the picture, hope you can see it.
http://imageshack.us/content_round.php?page=done&l=img204/7104/pcrgel.png&via=mupload&newlp=1

Generally, I feel your PCR mastermix looks ok, no sign of DNA overloading from gel, no primer dimmer.

I assume you are using SM0313 GeneRuler™ 1 kb DNA Ladder(Fermentas). Your problem is there are smearing and unspecific bands above your 550bp band.


I suggest to raise your annealing temperature to 60C(for 30s) and decrease your extension time to 72C(for 30s).
Alternatively, try a gradient PCR as well to see if it helps.

Just my 2 cents and let us know your new result. Good luck.

Adrian.

adrian kohsf on Tue May 24 16:33:38 2011 said:

Hi Adrian,

I tried a test under your suggestion. http://imageshack.us/content_round.php?page=done&l=img5/7049/pcrmay242011.png&via=mupload&newlp=1

It did work, but the higher annealing temperature leads to low concentration of PCR product, you can see from the Gel, the band is weak( 8uL of product was loaded on the Gel) and there is also one non-specific band above the 550bp band. I need to do DGGE next, I wonder whether the low concentration will work in DGGE or not.

Thank you so much for your advice.

Holly

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

adrian kohsf on Wed May 25 03:31:46 2011 said:

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