E5300 FAQs for ProtoScript® AMV LongAmp® Taq RT-PCR Kit, RT-PCR and qRT-PCR, Intl, NEB
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ProtoScript® AMV LongAmp® Taq RT-PCR Kit FAQ

Q1: Why am I getting a low yield of cDNA?
Q2: Why do I see products of the wrong size?
Q3: Why do I have a low yield of PCR product?

Q1: Why am I getting a low yield of cDNA?

A1: There are several possibilities:
  1. Check the integrity of the RNA by denaturing agarose gel electrophoresis (2). 
    RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides (2).
  2. Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases (2).
  3. Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN.
  4. Use sufficient amount of RNA.



Q2: Why do I see products of the wrong size?

A2: These could be several reasons:
  1. Always do a negative control reaction in the absence of reverse transcriptase.
  2. In cases where the RNA sample is contaminated with genomic DNA, treat with DNase I before cDNA synthesis (5).
  3. Design primers spanning an exon-exon boundary.



Q3: Why do I have a low yield of PCR product?

A3: There are several things that may improve yields:
  1. Check the primer design using computer software.
  2. Optimize the annealing temperature in a 1-2°C step.
  3. A primer concentration of 0.2 μM is satisfactory for most PCR reactions. However, sensitivity and yield of RT-PCR reactions can be improved by increasing the primer concentration to above 0.5 μM. Lower primer concentration between 0.07 μM to 0.2 μM may improve specificity.
  4. Increase cycling numbers up to 45 cycles.
  5. Do a manual hot-start.
  6. Use thin-wall 0.2 ml PCR tubes.
  7. Try a touch-down PCR protocol (4).