Hi there,

I would just add one more control :

If you have already successfully amplified your genes from cDNA, you should check your primer sets in a PCR using the amplified fragments as templates.

Hi Megha,

I had the same situations before, my suggestion to you is to set one parameter (temp.) constant and standardize cycles and time at the same time and vice verse. I had many combinations to try and get what I want.

As you mentioned two trials would not be enough. Try more samples.check this link if it helps.

http://www.mobio.com/blog/2011/03/01/a-quick-guide-for-troubleshooting-problems-with-pcr-amplification/comment-page-2/

Good luck

Hello,

I had the same problem before... Possible reason is that your cDNA is degraded or less amount of cDNA.

First of all, make cDNA fresh and keep it in -80'C, better do not store cDNA at -20'C.  Do not freeze and thaw cDNA more than 3 times.

Secondly, add more amount of cDNA..

Good Luck!

hi, 

the main problem in PCR amplification of cDNA are related to:

quality of water ( need to keep frozen , because chance pH). quality of cDNA can be contamination, quality of buffer, make reaction always in ice, make gradient study to have a range of temperature of anneling. Check primer concentration running a acrilamide gel at 6%

good look

Thanks everyone for all your suggestions.

I used primers to amplify actin from cDNA of all aliquotes. It was found that the primers had got degraded. And, 2 alliquotes of cDNA were degraded.

I am gonna set up with the primers of gene of interest. And, get back to you all.

Good. take new set of primers and prepare fresh cDNA and try it. 

I agree with Kaushal's three step suggestion with addition of Ajit suggestion to try fresh cDAN.

Hello Everyone. I did used fresh cDNA and tried to amplify the gene of interest with new set of primers along with positive control. I could amplify it.

My pcr is working on & off. 

I use an enzyme master mix. Please, suggest me. why, my pcr sometimes works and not works?