Hi

If you already got the amplification before with the same conditions, and now you dont get the same results problem might be the really concentration of DNA ...when the DNA sample stays for long time in plastic tube it tend to bind on the surface of the tube. So try to run agarose gel to see if you really have (probably you are putting less than what you think).

Goodluck

cDNA is very labile because it is single stranded. When you say you are using the same cDNA sample then if this has been freeze-thawed or kept in the fridge for long periods then it may have degraded. I typically freeze aliquots of cDNA such that each can be used a single time. You may therefore need to produce more cDNA from your RNA extract (aliquot) and repeat your PCR on this new preparation.

Hi Shobhita,

I aggree with Peter. You can use Low Bind tubes, to pervent DNA attaching to the plastic wall of the tube. E.g. MAXYMum Recovery tubes (Axygen) (PCR or 1,5 ml)

In the terms of DNA amount, I advise you to use the same DNA amount per reaction to reduce the variation come from template amount.

To optimise your reaction I suggest, to do a touchdown PCR. If you can use PCR machine available with this feature. For the annealing temperature just set the highest temp for exmple 65-70 °C. Set the PCR to decrease the annealing temp by 0.5 °C after every cycle down to the calculated annealing temp of your primers. For example 55 °C (lowest temp). Run the PCR on this temp for the following calculated cycle number. For example: (total cycle number)-(numbers of decreasing temp cycles) = (remaining cycle number). E.g.: 40 cycles - 20 cycles = 20 cycles at 55 °C. This technique not as elegant as gradient PCR, but it can be usefull. Unf you won't know the ideal annealing temp like in grad PCR, but it will maximize the amount of the specific product. It's much more ellegant way to find the best annealing temp empirically to just run a gradient PCR if you available an machine with his feature.

Cheers,

Tamas

You should check the cDNA samples with agarose gel to see if your cDNA is still there is or not. That can give you an idea. After that, you can use qPCR to check some reference-housekeeping- genes.

Hi Shobhita,

1) What primers you used for making cDNA?

If it was Oligo-dT then using same cDNA you may check any internal control gene such as 18S rRNA or tubulin etc. expression. That will clear first doubt whether cDNA is intact or not.

2) Synthesize fresh cDNA using Oligo-dT if your gene is protein-coding otherwise use gene specific reverse primer.

3) Check your primers on gel and change PCR components.

Checking of cDNA on Agarose gel will not lead to anything because cDNA in both condition, intact and degrade, appears as smear due to RNA presence.

good luck!

Thank you all for suggestions

i made fresh cDNA and also checked on gel, then tried @ 58, 60, 62 and 64 temperature but i am getting only sharp non specific bands.

to make cDNA i generally use BIORAD cDNA synthesis kit which work quite well.

What is your primer there for cDNA synthesis?