This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
, No Gclids
PCR Troubleshooting
Overview
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].
Troubleshooting Guides
Download troubleshooting handbooks for IHC, Western blot, and ELISA for FREE.
Troubleshooting GuidesDNA 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 |
Do you need a miniprep kit?
Boster has a convenient Miniprep Kit For Plasmid DNA Extraction And Purification.
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 |
Looking for Master Mix?
Boster has convenient PCR Master Mixes. Save your time and reduce contamination with our pre-mixed formulations!
Related Pages
PCR Protocols
Learn stepwise PCR protocols from reagent preparation to data analysis. Check out their differences to learn how they compare and their advantages and disadvantage.
See MorePCR Troubleshooting Guide Download Page
This guide will teach you everything you need to become a PCR expert, including a critical review of PCR and molecular biology principles, protocols, all-in-one FAQs, and more. Optimized Protocols Need protocols for DNA and RNA extraction and purific...
See MorePCR and Molecular Biology Fundamental Principles
The DNA strand used to produce the complementary strand is referred to as the template strand, which is used to synthesize complementary strands of each parental strand. These molecules are responsible for giving information to cells of each organism...
See MorePCR Sample Preparation
PCR sample preparation is very crucial for reliable results. It includes accurate extraction and purification of DNA or RNA to the library preparation and screening.
See MoreChIP Troubleshooting Guide
protocol and troubleshooting chromatin immunoprecipitation chip troubleshooting guide ChIP Troubleshooting Guide|Chromatin Immunoprecipitation Troubleshooting
See MorePCR Technical Resource Center
These molecular biology techniques have various broad and useful applications in our scientific community, so understanding the fundamental principles will be useful in your journey. Manipulation and investigation of genetic material is done through ...
See MoreImmunohistochemistry (IHC) Troubleshooting Guide
This guide provides a thorough list of Immunohistochemistry troubleshooting tips, including weak staining, high background, nonspecific staining among others. Learn how to take control of your IHC process.
See MoreFlow Cytometry (FACS) Troubleshooting Guide
Boster Bio is an antibody company and supplier. Read more about our troubleshooting tips for flow cytometry (FACS) experiments. Learn tips on how to resolve issues such as high background, and weak or no signal
See More