Synthesis of Large Size of cDNA
How to synthesize the large size of cDNA form viral positive RNA ?
1. The first thing to clarify is the positive control for PCR that you are talking about is it using the primer pairs for your target gene? If it is, then that means your PCR condition and primers work. If you are just using another 3kb gene, the positive control you got is only telling you that your PCR condition such as your enzyme, dNTPs or buffer are working fine, it still can’t tell you that is annealing temperature a problem.
2. “I also tried to amplify the Beta-actin gene from insect together with the virus gene, but no band of beta-actin gene as well.” I think beta-actin gene belongs to your host cell while virus gene belongs to the virus. When I extract viral RNA, I don’t lyse the host cells and viruses together, I will just take the viral supernatant and extract the RNA.
3. Yes, I think your approach is right, using a small fragment to detect your virus. If there is nothing in the beginning, then no virus no viral RNA. Alternatively, you can observe is there any CPE occurring when you grow your virus provide that your virus can produce CPE.
Save time and excel in your essays and homework. Hire an essay writer for the best price for the top-notch grade you deserve.