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Monday, May 27, 2013

Troubleshooting your PCR Reaction


This is a guest post from one of the blog readers.

In a PCR reaction various things can go wrong, So interpretation and troubleshooting a PCR reaction is important in order to further proceed with the PCR results:

qPCR amplification curve, pcr amplification


Possible Issues


1. No Ct Value / Signal:
This is a common problem faced by many researchers in the lab. PCR reaction is setup and at the end of PCR reaction No reult (No Ct / No Signal), waste of reagents and time.


Possible cause:


  • Problem with PCR mastermix.
  • Forgot to add template DNA
  • Template DNA used is degraded.


Solution:


  • Perform PCR reaction in duplicate or Triplicate.
  • Make sure template DNA is added.
  • Keep a Positive Control and Negative control along with Samples.
  • Re-do the reaction with fresh set of reagent stocks.


2. Evaporation:
Modern PCR mchines will not be having this issue, but the older ones do have, evaporation occurs during the temparature cycling in a PCR reaction, as the temperature can reach upto 95oC causing the mastermx to evaporate from the PCR tube.


Solution:


  • Use mineral oil, Silicone oil or Paraffin beads to prevent evaporation..


3. Poor or Low Signal in real time PCR:
Poor signal can be due to the following reasons


  • Degraded Probe.
  • Probe concentration sub-optimal.
  • Tempalte DNA having Salts (carried over from extraction).
  • Higher concentration of Template DNA.


Solution:


  • Use Fresh aliquot of Probe
  • Optimize Probe concentration
  • Re do PCR reaction by diluting template DNA.


4. Amplification in Negative Control:
The negative control used in a PCR reaction is to verify the reaction has any contamination. Negtive control used will have no template DNA, so nothing should amplify. If something does amplify, it is due to contamination in the reagents, and indicates that the bands in the other samples are likely contaminated as well.
The negative control would be water in place of DNA.
The PCR experiment is not valid without a Negative control.


Causes:
  • Negative control is contaminated with template DNA.
  • PCR Buffers and reagents are contaminated.
  • Primer Dimer formation (Rare chance)


Solution:


  • Prepare fresh set of reagents.
  • Clean working areas, Pipettes with hypo.
  • Use gloves while working.
  • Have separate areas for mastermix preparation, Sample addition and Post PCR analysis.


5.Variation in PCR duplicates


Cause:


  • Inaccurate Pipetting


Solution:


  • Pipette calibration.
  • Follow Proper Pipetting technique.


6. Poor PCR Efficiency
Poor PCR efficiency can be due to many factors in a PCR reaction to list a few


Possible Cause


  • Inhibitors
  • Sub-optimal Primer Probe ratio
  • MgCl2 Concentration not optimal
  • dNTP concentration
  • Inacurate Pipetting
  • Taq Concentration
  • Problem with annealing temparature


Solution:


  • Optimize PCR Buffer, Mgcl2, dNTPs, Taq
  • Try with different annealing temperatures.
  • Pipette calibration.


PCR Product analysis on Agarose gel

In End point PCR or conventional PCR running an Agarose gel is the only way to analyze the PCR product.Even in the initial optimization stages of real time PCR, Agarose gel is performed to analyze the PCR amplification. What all can go wrong when you run a PCR product on Agarose gel.

PCR Product smear gel





  • Smeared Band

Due to higher concentration of DNA / Presence of impurities, this can be solved by diluting the sample before loading.


  • No or Faint Band
Due to No amplification or Poor amplification - this can be solved by optimizing PCR buffers and primers for the particular target.


  • Multiple Bands

Due to primer dimer formation - This can be solved by :


  1. Using Hot Start Taq Polymerase
  2. Optimizing MgCl2, KCL concentration
  3. Primer Specificity
  4. Designing new Primer set with optimum GC content.