Hi,
Here is my PCR Reaction The primers I used were both 10µM.
I added 2µl template cDNA
and diluted to 10µl total with water.
The cycle used was as follows:
95c | 5'
95c | 30"
62c | 30"
72c | 1'
72c | 10'
I ran the products on 1%agarose gel ,100v and for 1h with the 1kb ladder.
The product was approximately 900bp and as you can see,
Both of test and control bands are opaque(I don’t know its exact technical term,maybe smear) and include nonspecific bands .
How to troubleshoot?
And how will the problem be resolved?
Any suggestion or comment in this regard will be helpful
Thanks
Niloufar
1. Decrease the DNA concentration ~30-50 ng is more than enough.
2. Increase the Agarose concentration up to 1.5 % run gel in 50 V
3. Try to increase Denaturation and annealing time to 45 secs
4. Put Gradient as 55-62 °C
Go slow!
I guess you are looking for one of few bands? you gel was obviously ran too quickly!
If you still have of this PCR reaction, run a slow overnight gel, you'll see better.
What kind of DNA are you actually studying?
Thanks dear Benjamin.
Our sample is human cDNA
In order to running an overnight gel, what voltage and percentage of gel do you suggest?
Thanks
I suggest to use 76°C and not 72°C.
do you try a gradient of the Tm annealing?
Thanks dear Giorgia
Yes I did it in TA 60 -61-62
T 61 was the best ,
Band of interest was clearer,
but nonspecific bands still remained.
@giorgiapertile
Try lower Tm than 60 °C and try to prepare a dilution of you cDNA at 1:10 and add to the mix 1 ul.
More time the smear means than you put so much DNA/cDNA in the mix.
1. Decrease the DNA concentration ~30-50 ng is more than enough.
2. Increase the Agarose concentration up to 1.5 % run gel in 50 V
3. Try to increase Denaturation and annealing time to 45 secs
4. Put Gradient as 55-62 °C
Hi Niloufar,
the 10 µM primer concentration seems excessive to me (not sure whether that could cause the smear - never tried that high conc). Usually, in "standard" PCRs the primer concentrations vary somewhere between 0.05 to 1.0 µM. Of course, that depends on the chemical system you use. Usually, the provider of the polymerase suggests a range of concentrations - did you check those? Also, as mentioned above, it might be too much DNA. We usually use 1 ng/µl DNA in "standard" PCRs. As for unspecific bands, you may also try to lower the Mg2+ concentration in order to increase stringency (additionally to the suggested highering of the annealing temperature).
Regards,
Klaus
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