funny, I am having a similar problem at the moment..

is your marker lane looking okay? or do you get a smear there too?

Has your product the right size?

I got some improvements with changing my loading dye and the running buffers..

yes my ladder is fine no worries about that

There are a few other details that might help figure out what the problem is. Which polymerase are you using? What is the expected size of your product? (it would help if you included the marker) Do the primers contain restriction sites & if so, what is the complementary sequence to your target?

I am using Taq polymerase

product size is 1182 bp

the primers are for LIC (Ligation Independent Cloning) which contain no restriction sites

When using IDT (http://eu.idtdna.com/calc/analyzer) to analyze, the Tm for Forward primer is 65.9oC, and for Reverse primer is 70.2oC. The Tm values you listed above are a little bit off. Did you only calculate 'partial' of the primes for their Tm values?

yes that is one mistake I have done and did correct it in my subsequent pcr runs (just yesterday) by adjusting the annealing temperature to a gradient from 60 to 70.... and thats the pic....

Hi Milsee,

1. Have you checked your DNA template on a gel, to see if the quality of your genomic DNA is good? If the gDNA is good, there should be a band.

2. Include a positive control (or an internal control). This will tell you that your PCR machine, PCR reaction setting up are not the source of problem.

Internal control: a specific gene present in this specific organism genome and can be always amplified if the PCR conditions are right. In tobacco, I use EF1-alpha as internal control.

I have used beta actin as internal control and I do get a band. template is cDNA. as pointed out by Zofiya, the reverse primer does make hairpin structure. can hotstart help solve this problem????

Hi Milsee,

1. In some RT-PCR instruction, PCR cycles are suggested from 30x up to 40x depending on your cDNA template. With low amount cDNA template, you may want to increase your PCR cycles. So, you can do one PCR to check.

2. You can also try using robust DNA polymerase such as Ex-Taq, LA-Taq (Takara) [or other brand names of robust DNA polymerase] to increase the PCR efficiency.

3. Add 2-4% of DMSO in your PCR reaction if you have G-C rich template or secondary structure to increase the specificity and efficiency.

4. The best way to find out your question about haipin/hotstart is to do an experiment to find out.

Best,

hi Yuan

thanks for all the suggestions

I will try those and get back to you, hope fully with a positive amplification result :-)

hello all

Finally I have got the amplification for my gene.....

As always with PCR, if your primers are good it just works outfine

Problem was with my primer set

Solved and got the results

Thank you all for all the suggestions, it helped a lot

Hi Milsee,

Good job, and thank you for updating us. Just curious, you re-designed the primers and what criteria of the new primers have you changed and led to the success of your PCR?

Hi,

It was a mistake that i did when I designed the reveres primer, I did not reverse it in the 5'to3' direction when I gave it for synthesis.....silly mistake.....hehe

Well, mistake happens. Glad you caught it. Good luck on the rest of your experiments.