Troubleshooting guides

Troubleshooting guides

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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 [email protected]

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

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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

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