I have 3 different mammalian expression vectors carrying one/two HIV genes. I transfected them to African Green Monkey CV-1 cells. 72 hours after transfection, I did total RNA isolation from each. My RNAs are high quality. Then, I did first strand cDNA synthesis using specific primers. I used these cDNAs to run real time PCR. My PCR cycles are 95 C-10min (1X), 95 C-15sec(40X), 62 C-30 sec(40X), 72 C-30 sec(40X), 95 C-1 min(1X), 55 C-1 min(1X), 55 C-30 sec(81X). I used SYBR Green q-PCR master mix. It is recommended to use 95 C-10min (1X), 95 C-15sec(40X), 62 C-30 sec(40X), 72 C-30 sec(40X) cycles. And also, one of my professor told met hat he adds 95 C-1 min(1X), 55 C-1 min(1X), 55 C-30 sec(81X) cycles after his cycles to see melting of primers. Then, I realized that there are amplifications in an expected size even in the constructs which don’t have the gene. I thought that it could be due to contamination. I changed all the stuff: my primers, master mix, water and I run another 2 PCRs using these cycles. At first, I used my new cDNAs, second I used diluted vectors. But again, it showed same results. There are also strong primer dimers in no template controls even I used only 10 mM for 25 ul reaction. Then, I run another PCR with a template which is totally different from my constructs and don’t have any of my genes, but there are still amplification. It seems there is a primer problem, not contamination, but I cannot solve it. Any help would be appreciated.
Thanks in advance..
Burcu

Your primer concentration sounds high (mM). I don't understand why you do 81 cycles of 30 seconds extension at the end either.

What is the expected band size? Have you ever run the product on a gel? After 40 cycles, it is not unusual to have some negative controls coming up. Do the signals in your cDNA lacking samples come up early or right at the end?

1.As I mentioned before, one of my professor told me that it is useful to add these cycles after it, if you want to see your primers' melting.
2.I use 0,5 uM primer in qPCR cycles. But when I had a problem with qPCR results, I repeated all using PCR master mix. Therefore, I used 10 mM from each primers.
3. My expected band sizes varies 90-150 bp.
4. I always run my products on a gel.
5.I have to check it again. I don't know much about qPCR data analysis. At first, it seemed okey, but the last repeats there were problems. But I also have problems when I used plasmid as a template instead of cDNA. It is also interesting.

1. Yeah, I get a product in an expected size in reactions that contain no cDNA, no template or template which is totally different from my gene sequences??
2. I did DNase 1 digestion.
3. I thought that it would be more specific to use gene specific primers to make cDNAs, but this time I will use oligo dTs, I have realized it.
4. Sorry for making you misunderstood me. I want to clerify the primer amounts that I used, because I didn't write final concentrations, you misunderstood me. If I'm using PCR master mix, I'm using 1 uM primer. If using qPCR master mix, I'm using 0,5 uM as indicated in the protocols. Big question mark???
5. I'm sure for my annealing temperature, because I made optimization before.
Thanks for all your help..

sorry but do you clean properly the pippetes before the qPcr? The size of the qPCR are ussually small and is easy then contamine your solutions througt the pippete...

It was the only thing I haven't thought before. I will try all the reactions with new pipettor, thanks.

Use filter tips on the pipets. It costs more but it helped me a ton with this problem. We use the fisher brand or ART, but I like the ones from Bioexpress the best because they stay on the pipetman better.

rb