9 replies to this topic

[vfpp]

Hi,
I have been having a serious problem to determine the genotype of mice because I can not recognize the bands - between 200 and 350bp. I am extracting the genomic DNA from mouse tails using the high salt method and after that I perform a simple PCR using the PCR kit from Qiagen. The procedure was working fine until recently, when I started to have trouble to detect the PCR product (bands should appear at 200 and/or 350bp) - please, see picture attached. As you can see by the attached picture, I am getting a very strong unespecific band close to 100bp and that's it! I purchased new primers, hoping to have the problem fixed but it did not help me... Also, I prepared new reagents (i.e.: dNTP mix and TAE) to see if it could help but again, it did not work. What should I do?? Does anyone of you know what should be going on?

[hobglobin]

Are you sure that the strong unspecific band are not primer dimers? Not sure as I don't know the DNA ladder band sizes...
You should check DNA quality (size, amount) and also try out a fresh aliquot of primers....

[Trof]

Yep, looks like dimers. Where is your negative control?

[vfpp]

First of all, thanks a lot for your comments on my topic.

The DNA ladder bands are 100bp, 200bp, 300bp, and so on...
As for the primers, I just tryed a fresh aliquot and I got the same problem... Do you think there is a problem with the method that I am using to digets the mouse genomic DNA?? For the DNA isolation, I am using the following protocol:
BASIC DIGESTION BUFFER:

• 25mM NaOH
  
• 0.2mM disodium EDTA
  
• Dissolve with sterile H2O

PROCEDURE:

• Cut and place a small piece of the tail (1mm) in a small tube
  
• Add 100μl of Basic Digestion Buffer; spin
  
• Heat at 95˚ for 60 min

[Trof]

If you make a reaction mix same as for your samples, but replace DNA with water, do you get this band as well?

[vfpp]

Yeah... That`s my next step - It was really stupid but I forgot to include a negative control...

[Gizmodo]

We use a similar method of digesting our tails, but we use a neutralization reagent (ddH20 and Tris-HCl) after the 95 deg C heating process. If you are not quenching the tails with an acid/buffer, the strong NaOH could be inhibiting your reactions somehow. Though, I would agree, it does look like you have primer dimers of some sort. So, perhaps my suggestion is useless.

[vfpp]

Thank you very much for your suggestion - I am going to try to quench the tails.

As for the negative control... the strong "unespecific band" at 100bp also shows up on the negative control (!). So, I don`t believe that this is a case of primer dimer...

[hobglobin]

well you also added primers there, so why not? did you design the primers yourself newly or are they well known ones that worked for a long time well? if the first is true you should check the primer properties

[vfpp]

The primers were previously designed and oddly enough were working very well until a couple of months ago...