Molecular Biology and PCR Experimental Troubleshooting

    The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from molecular biology experimental techniques. We at Boster Bio are committed to helping our customers get better results. While the troubleshooting guide below covers a multitude of problems encountered while performing in the lab, we do not expect it to be the exclusive solution to any problems during your specific experiment. We hope that you will find the information beneficial to you and useful as a reference guide in troubleshooting any problems you may encounter. If you ever need more assistance with your experiments, please contact the Boster Support Team by email at support@bosterbio.com

    Jump to:

    1. DNA and RNA extraction
    2. PCR and qRT-PCR

     

    DNA and RNA extraction

    Problem

    Possible Solutions


    Low yields

    Increase sample volume

    Increase lysis time or add enzymatic lysis step

    Increase lysis time in 10 min

    Make sure that vortex and resuspension steps allow a good homogenization

    Suspend DNA or RNA in less volume

    Salt contamination

    Repeat extraction protocol from precipitation process

    Protein contamination

    Increase lysis time or add enzymatic lysis step

    >>Did you know Boster has a convenient Miniprep Kit For Plasmid DNA Extraction And Purification?

    Check Now

     

    PCR and qRT-PCR

    Problem

    Possible Solutions





    No amplification

    Perform a temperature gradient PCR

    Make new primer work solution

    Increase template concentration

    Decrease Tm  temperature

    Increase cDNA concentration

    Check DNA template quality in Nanodrop

    Verify time and temperature settings

    Use new template





    Non-specific amplification

    Increase Tm temperature

    Avoid self-complementary sequences within primers

    Avoid stretches of 4 or more of the same nucleotide or dinucleotide repeats

    Lower primer concentration

    Follow general rules of primer design

    Decrease the number of cycles

     

    Amplification in negative control

    Use new reagents, namely buffer and polymerase

    “Homemade” polymerases usually contain genetic contaminants. Try a commercial polymerase instead.

    Make sure to use sterile tips

    Low yields of PCR product

    Increase number of cycles by 10

     

    High quantification in standards

    Use new diluted standards

    Check pipettes calibration

     

    High variability in replicates

    Verify pipettes calibration

    Use fresh diluted standards

     

    Erratic curves

    Calibrate optics of the system

    Repeat with rox-normalisation dye

    Bands and smear are very intense

    Reduce the number of cycles

     

    Incorrect product size

    Look for additional primer complementary sequences in the template

    Increase Tm temperature

    Make new primer work solutions

    >>Did you know Boster has a convenient PCR Master Mix? Save your time and reduce contamination with our pre-mixed formulation!

    Check Now