FAQs about reverse transcriptase
How can I design RNA-specific primers?
You can use one of the free primer design tools in the internet, such as primer-blast on this website: www.ncbi.nlm.nih.gov/tools/primer-blast/ >. Ensure, that you select the option: "primers must span an exon-exon junction". This is useful for limiting the amplification only to mRNA, as HotScriptase RT Poylmerase will amplify from any nucleic acid target consisting of both RNA or DNA.
I am only getting primer dimers. How can I establish my RT-PCR?
- Keep all reagents during PCR setup on ice.
- Programme the PCR protocol on your cycler first, before you setup the PCR mix and if applicable preheat the lid of your cycler.
- When you do a new PCR for the first time, start with our standard protocol and recommended reaction setup concentrations.
- Run a temperature gradient to identify the best temperature yielding in the cleanest product.
- Use a "multiple primer analyzer" to test your primer sequences for self-complementarity, secondary structures and primer cross annealing.
My negative control reaction with Taq DNA polymerase is yielding a specific amplificate - what can I do?
- Perform a reaction in the absence of any template, e.g. just MQ-water instead. If you are still getting a specific amplificate, you most likely have a contamination problem. Shortest solution is to start over with fresh samples and fresh reagents.
- Analyse your primer sequences and perform a primer BLAST to ensure, that your primers are only RNA-specific, for example here:
http://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/. In case they are not RNA-specific, DNAse digest your samples in order to remove any traces of DNA and try it again.
- Alternatively, design new primers, which are binding on exon-exon junctions in order to avoid amplification of residual DNA.