I am doing a rtPCR to detect the watermelon mosaic virus (WMV). My set of primers are:
- WMV primer forward: 5'-TNGARAATTTGGATGYAGG-3'
- WMV primer reverse: 5'-CTGCGGTGGACCCATACC -3'
both of which at the concentration of 10 microMolar.
The first step of my rtPCR reaction was looking for a positive control.
I did the rtPCR against to a field sample (In the NGS results showed a positive against to WMV) and two greenhouse samples (the first one of my greenhouse samples was inoculated with WMV and the second one it was not).
These samples were extracted with good concentrations and integrity. After that, I did the rtPCR with rt enzyme (from Affinity company). Following the next steps:
- I added 2000 ng of my RNA samples (in different tubes; one of them to one greenhouse sample, another tube for the remaining green house sample and the latest tube with the field sample) with Reverse primer (2 ul) and H2O (until get 12.5 ul).
1.2. I incubated them 65ºC 5' and 25ºc 10'.
2.1. I added the next volumens for 1 sample: 2ul of my buffer, 2ul of my DTT, 2ul of my DnTps, 0.5 ul RNAsa block and 1 ul of Rt enzyme.
2.2. I vortex them and I incubated them 1 hour 50ºC and 10' 70ºC.
3.1. The PCR reaction was performed with the takara polymerase enzyme. I added the next volumen per one tube: 2.5 ul of takara buffer, 0.5 ul forward primer, 0.5 ul and primer reverse, 0.5 ul DNTPS, 0.25 ul of taq polymerase and 15.75 ul of watter. All these volume 20 ul I added 5 microlites of my rt samples (2.2 point). I did this process with my remaining two samples.
3.2. Finally I incubated them 98ºC 5', then 35 cicles of 98ºC 30'', 56ºC 30'' and 72ºC 1'. out of the 35 cicles the temperature goes to 72ºC 10'.
I run the gel and I got primer dimmer. What I am doing wrong? All help will be very useful.
Note: These protocol was following by a labmate who got good results with the same field sample. Now I am following the protocol that was written in his notebook.