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PCR malaria diagnosis nested PCR smear and non specific bands - (Mar/27/2012 )

Hola! I am really struggling with a PCR and I will definitely appreciate your input.

So this is a nested PCR with the following conditions:

First reaction

94C 5min

94 1 min
60 2 min
72 2min 35 ciclos

72 10 min


Second reaction, the same as above, only the annealing temperature is 55

PCR conditions (the same for first and second reaction, onlly primers different and the amount of template, first reaction 2 ul DNA, second reaciton 3 ul PCR product)

Buffer 1 x
Hot start Promega GoTaq 1.25 U
MgCl2 2.5 mM
dNTPs 200 uM c/u
Primer 200 nM each


However, I am getting tooo many smears and non specific bands (Please see the file) I have tried different concentrations of primers withouth any luck. Can you please provide any suggestions you think I can do?

In the gel, you will see:

1 --MArker (50 bp) Novagen

2--Pfalciparum 1 parasite/ul
3--Pfalciparum 2 parasite/ul
4--Pfalciparum 3 parasite/ul
5-Pfalciparum 4 parasite/ul
6-Pfalciparum 3d7 1:20 (conttrol positive)
7..Control negative Pvivax
8--Control negative Water first reaction
9--Control negative Water second reaction

Expected size: 205 bp

By the way, just tonight I have tried changing the second reaction by decreasing to 30 sec the three temperatures and only 30 cycles, still waiting for that result.
Thank you very very much.
Maria Eugenia
Attached Image

-maruca8-

Many possibilities and questions...some thoughts:
Why a nested PCR?
Did you check the results of the first PCR alone?
For 205 bp 2min extension time is much too long...15-30 s should be enough (or even less)
Reducing Mg-concentration and cycling numbers might improve specificity
less Taq (0.5 -1 U is also enough usually, reduces possible unwanted amplifications and saves money too)
Try gradient PCR to optimise annealing temperature., i.e. a higher Ta might work also
Not sure if less cycles in the first part of the PCR might help, too

-hobglobin-

Thank you very much hobglobin, I have today better results after changing parameters in the second reaction, I used 30 seg for denaturation, annealing and extension. I also used less Taq, 0.5 U worked very well.

The only problem I have today is that after thinking everything was working finally fine, I run 50 samples and the results were a little weak compared to before, I think that might be for the volume that I prepared, or maybe too much time I took for preparing everything.

Will work tomorrow with less samples, but thank you veyr much for suggestions, they definitely made a difference and I really appreciate it.
Thanks!

-maruca8-

Just to add to hobgoblin, using 3 ul of 35-cycle PCR product into second reaction seems quite a lot. Usually the product from first round is diluted 10-100 times, when used for second round (1 ul).

-Trof-