Common questions and answers of the PCR machine

Common questions and answers of the PCR machine

1. Very low cDNA yield

possible reason:

*RNA template quality is low

*The mRNA concentration is overestimated

*There is a reverse transcriptase inhibitor or insufficient amount of reverse transcriptase in the reaction system

*Isotope Phosphorus 32 expired

*The reaction volume is too large, should not exceed 50μl

2. The amplified product has no band or very shallow band during electrophoresis analysis

*The most common reason is that your reaction system is PCR reaction system instead of RT-PCR reaction system

*Related to the total amount and purity of RNA at the beginning of the reaction

*It is recommended to add control RNA to the experiment

*When performing PCR amplification, the content of the reaction product of the first strand in the total reaction system should not exceed 1/10

*It is recommended to use Oligo (dT) or random primers instead of gene-specific primers (GSP) for first-strand synthesis. Due to the secondary structure of the RNA template, such as circular results, it is possible that GSP cannot anneal to the template; or SSII reverse transcriptase cannot effectively extend from this primer.

*The target mRNA contains a strong transcription stop site, you can try the following methods to solve it:

a. Increase the reaction temperature of the first chain to 50°C.

b. Use random hexamers instead of Oligo (dT) for the first chain reaction.

3.Produce non-specific bands

*Use RT negative control to detect whether it is contaminated with genomic DNA. If the PCR result of the RT negative control also shows the same band, the sample needs to be reprocessed with DNase I.

*In a PCR reaction, non-specific initial amplification will lead to non-specific results. Annealing at a temperature lower than the primer Tm 2 to 5 ℃, reducing the amount of magnesium ions or target DNA will reduce the generation of non-specific results.

*Due to the different methods of mRNA shearing, the selection of primers will lead to different RT-PCR results.

4. Produce smear bands

*The content of the first strand product in the PCR reaction system is too high

*Reduce the amount of primers

*Optimize PCR reaction conditions/reduce PCR cycles

*When DNase is used to treat RNA samples contaminated with DNA, the oligonucleotide fragments produced will produce non-specific amplification, which will generally appear as a diffuse background.

5. Produce large-molecular-weight diffuse bands

*In most cases, it is caused by non-specific initiation and extension caused by too low annealing temperature

*For long fragment PCR, it is recommended to dilute the cDNA concentration in the reaction system to 1:10 (or 1:100-1:200)

6. In the absence of reverse transcriptase, control RNA to obtain amplification results

*Usually caused by trace amounts of DNA in the control RNA. Because it is impossible to eliminate all DNA templates during in vitro transcription. It is recommended that the first strand cDNA be diluted 1:10, 1:100, 1:1000 times to eliminate the effects of DNA contamination.

*It may be a band of primer dimer

7.Amplification product stays in the sample well

*It is possible that the amount of template is too high and the PCR result produces a high molecular weight DNA jelly. It is recommended to dilute the results of the first strand by at least 100 times before performing a second amplification.

*In addition, if the annealing temperature used in the second PCR is 5°C lower than the Tm value of the primer, the annealing temperature can be appropriately increased or a hot start can be performed to improve specificity.

8. What is the difference between SSⅢ and SSⅡ?

*With higher thermal stability (up to 50°C)

*Has a longer half-life (up to 220 minutes)

*No inhibition to PCR

*Dry ice transportation

*Tdt activity is lower

9. Why do some people prefer to use SSⅢ instead of ThermoScript?

If ThermoScript is not stored properly, it will cause a rapid decrease in activity, while SSⅢ is more stable.

10. Why use gene-specific primers (GSP)?

GSP is best when amplifying low-abundance transcripts. OligodT primers are recommended for reverse transcription of high-quality RNA and full-length transcripts; random primers are used for reverse transcription of mRNA fragments.

11. Under what circumstances need to use RNase H?

When RNA/DNA hybrids cannot be denatured normally in the first round of PCR