Why do I not see the Internal Control Gene at step 1 PCR?

Is there any site or anynone here where i can get In house real PCR Protocols for HIV DNA diagnosis, HPV, HBV viral load, HIV viral load, Breast cancer and lymphoma PCRs

Thanks

Hi all! I have been trying to amplify the 5' UTRs if my gene using the 2nd generation kit from ROCHE. This kit uses an specific primer to generate cDNA and then you add a dATP tail at the 3' end of that cDNA, from that you perform a PCR using an anchor primer they provide and an specific primer and they give you the option of performing a nested PCR if you see no product or a smear in the first round.

The first time I tried this, I got truncated products (the adapter primer had bind to an internal part of the gene), so I decided to try it again (this time I made sure my RNA was good quality) but have been having problems. Last time I tried, I got a band (with lots of smear) from the first PCR round, but nothing when I tried to amplify using internal primers (nested PCR).

Has anyone has any experience using this kit or know what can be happening and why can I be not getting product from the nested if I got a product in the first round?

Thanks!

Hello,

Are there anyone can help me to do troubleshooting on my latest PCR to extracting DNA from AMF spores. I'm doing a nested PCR using a reference which has used GeoA2, Geo11 and AM1, NS31 primers. Initially, I followed all the steps same and running several times using different concentration of template I had got a good result. Unfortunately, just before send sequencing someone has dumped it. So I'm doing it again and again but all the results are being same like have my desired band in there as well as smears or primer dimers under my bands.

I tried to change new reagents expect primers, also reduced the annealing temperature and tried different dilution of templates

The picture below has shown my 2nd run and 3rd run. You can see from the first image, almost all the wells have shown the same sized bands and smears.

The second image, I'd chosen some of the previous pcr product and diluted them 10 and 50 times. It has show all primer dimers.

It's my first time doing DNA extraction from spores. Please, give me some suggestions.

Thank you!

The reference paper is here DOI: 10.1111/jam.13714

Good day house.

Please what is the function of the universal primer, Inosine, in a primer? I need to use the primer for PCR. If I don't include the Inosine in the primer as published what is the consequence?

While using primer blast to confirm my primers, I discovered that replacing Inosine with any of the four nucleotides, the primer blast gave me the set of organisms I am interested in. So, I am wondering rather than embed Inosine in the primer, why not replace it with any of the four nucleotides.

It is difficult for me to design a different forward primer for the second round PCR in nested PCR, so, I am wondering what will happen if I use the same forward primer that I used in the first round PCR? Will this still give me a more specific product or will amplify the nonspecific product.

I want to do MSP for InsR and slc2a4 gene. My template DNA comes from skeletal muscle and blood of diabetic Mus musculus. After DNA extraction I treat the DNA with bisulfite treatment. I'm still in optimization stage. Before MSP I do nested PCR to my bisulfite treated DNA. I tried to change primers concentration (10mM, 5mM, 2mM, 1mM, 0.5 mM 0.25 mM, 0.1 mM), used temperature gradient for annealing temperature (for both nested and MSP), and adding more template.

I uses PowerPol 2x PCR mix by abclonal as my mastermix. My friends tried to use the same DNA and they got their target band (Their target genes are FTO, KCNJ, PDK4, and PPARG). I set the PCR program exactly the same as the mastermix user manual suggestion. My mix contains 25 microliter PowerPol 2x PCR mix, 1 microliter primers (I have tried 10mM-0.1mM), 1-5 microliter of template(bisulfite treated DNA for nested PCR, Nested PCR product for MSP), and adding Nuclease Free Water until the final volume 50 microliter. What should I do to make my target band appear? The only things that appear in the gel are non-specific bands.

I used reported primer sets (first and nested PCR) and followed their protocol.

I'm getting a similar result to the reported result. However, for the nested PCR, I'm getting two bands that are unlikely to reported one. The nested PCR product size is 524 bp only.

I'm considering whether the first PCR primer set (1,180 bp) would affect the multiple bands, or does the annealing temperature needs to be optimized?

In the figure around 524 bp is the target, but 780 bp doesn't know.

I have been getting a band in the second (nested) amplification of the negative from the initial PCR, but not in my negative control testing for contamination. Any ideas?

Information:

I am using a nested PCR for different Ehrlichia species in whole blood.

The initial PCR is an Ehrlichia screen using one set of primers. For 20 cycles.

The nested PCR's have specific primers based on the Ehrlichia species. For 30 cycles.

The initial negative controls have RNases free water, Taq polymerase, and the Ehrlichia screen primers.

When I run the nested, I take 1 uL from the initial negative (which is clean on the gel).

I've recently changed out my reagents, and the band is around 500 bp so not primer dimers.

Could be something specific in the initial primers but I only see it in the samples that should be negative and it's a pretty big piece to be related to the primers.

Thank you