Hi Alaa, please specify which polymerase enzyme you used? Depending on DNA polymerase enzyme your PCR condition can be improved.

Cycling Conditions for a Routine PCR is attached: Initial Denaturation 95°C (30 seconds): 1X cycle Denaturation: 95°C (15-30 seconds) Annealing: 45-68°C (15-60 seconds) Extension:68°C (1 minute per kb ) These 3 steps should be X25-30 cycles Followed by Final Extension at 72°C (5 minutes) Hold at 4°C or ∞

I am assuming you have a good quality genomic DNA to start with. It should look intact on agarose gel and have the A260/280 and A260/230 absorbance value around 1.5-2.1. Then, to proceed with PCR, you should at least know these two important information:

1) Expected size of your PCR product. If you are using universal primers, a simple google search would get you to the information you are looking for. PCR product size is related to the PCR extension time. A conventional Taq polymerase works at 60 sec/1 kb. For example, I am using 27F and 1492R primers for molecular identification of bacteria. The expected PCR product size is about 1400-1600 bp. So I use 1:30 min extension time in PCR.

2) Primer sequence for you to calculate annealing temperature (Ta). There are many online primer calculators available. My favourite is always the IDT OligoAnalyzer https://www.idtdna.com/calc/analyzer . First you will get the Tm for each primer. Then you can calculate the Ta by summing the Tm of both primers and divide it by 2.

For the rest of the PCR cycle, please refer the recommended parameters stated in the kit's manual.

Hi Alaa,

2 ideas, have you tested that the primers you're using blast on the right genome? and the second, are you sure to have pure H.pylori in your sample, and not a mix of species?

fred

Thanks for all

I solve this problem by increase DNA volume 10 microliter

And Bovine serum albumin as inhancer 1 microliter

Additon of MgCl 0.5 microliter

Also i increase cycle up to 35 cycle