Help with PCR

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jcallum
jcallum's picture
Help with PCR

Hi

My name is JC. I need help with PCR problem. Performed CDNA synthesis using Quanta Bioscience FLEXI.

Oligo dt primer (2ul) and Random primer (2ul) were used in the reaction. (the two primers were mixed together to create a master mix for 16 samples). Primer mix was left overnight.

Complete the CDNA synthesis next day and did PCR amplification with another specific set of primers for target site around 300bp.

50ul of pcr product was generated using 4ul of forward primer  and reverse primer, + 25ul of 2xpromega mix+5ul of CDNA and made it up to 50ul with nuclase free water.

Ran electrophoresis with 1.5% agarose gel + 2ul of ethidium bromide.

No pcr product formed but huge primer dimer at around 100bp in the gel?

Could the primers in CDNA anneal when mix together.

Or has the RNA degraded- but RNA was extracted and stored -80 using qiagen RNAesy kit set. Extracted and stored at the same day and use 2 weeks after.

HELP-need some advice. I can't figure it out

Ivan Delgado
Ivan Delgado's picture
 

 
Hi JC,

First of all I do not think it is a good idea to mix both oligo dt primers and random primers when synthesizing cDNA. I would suggest you use only one. In my experience random primers work very well. 

Second, I would not have left your primer mix overnight. When synthesizing cDNA, prepare all reagents the same day you run the reaction.

Third, it is unclear from your question if your cDNA synthesis or PCR amplification were successful. For the former, if you have a control PCR reaction that always works (PCR control reaction), you should use that to test that the cDNA synthesis was successful. One good way to do this is to run the PCR reaction both with the cDNA you just synthesized and a DNA control (genomic, plasmid, etc) that contains your amplicon. 

Hope this helps.

jcallum
jcallum's picture
Hi Ivan

Hi Ivan

Thank you so much for your reply. Yes it does make sense, i don't have a DNA control as to run a comparison. However, from my question- i think my CDNA synthesis did not work properly as i have use the primer mix that is left overnight.

Reason for that is because i repeated the PCR amplification using the same CDNA synthesis and all i got from the gel electrophoresis is a huge stain primer dimer mix at around 100bp.

I will try your method and if all fails i will repeat my CDNA synthesis and do my PCR with the forward and reverse primer again.

By the way, another question is when you mix agarose with ethidium bromide how long do you swirl it for to make a uniform gel picture.

Cheers

Ivan Delgado
Ivan Delgado's picture
 

 
Hi JC,

No problem.

Mixing agarose and ethidium bromide is something that should occur almost immediately. Just swirl the solution for a few seconds and it should be more than enough. 

RomeoLima
RomeoLima's picture
I would suggest to dilute

I would suggest to dilute your cDNA, and to use this dilution as a template for your PCR reaction (four fold or five is Ok) . Sometimes after the reverse transcription reaction, the final cDNA must be diluted in order to reduce the amount of possible contaminants and/or  inhibitors that could affect your PCR reaction. Beside the possitive control, I always use as a negative control a non reverse transcription (No-RT) control, just to be sure that your amplification is due to the presence of the original transcript  and no because of genomic DNA contaminating your RNA sample.  

!-)