Frequently Asked Questions: Polymerase Chain Reaction (PCR)

Are you currently doing, or planning on doing one of our PCR kits, and have questions? Here are the answers to our most frequently asked questions regarding PCR!

Frequently Asked Questions:

Q: After staining, I can see the bands from the ladder well but not the bands for the control and experimental samples.

A: If the ladder is visible, it means that the staining process was successful. This indicates that the issue stems from the PCR thermocycler, not the staining process or the pipetting of the primer and DNA components. 

• Check that you are using the correct/matching literature for your perishable components. We update literature often, and sometimes, the cycling parameters change.
• Check that the cycling program that is saved on the thermocycler matches the programing scheme in the literature. Or, check that the program was inputted correctly if it was done manually.
• If the program was inputted correctly, the issue could be from the cycling conditions. Watch the program go through a few cycles, and make sure that the cycler is able to reach all the high and low temperature points. If the cycler is unable to reach these temperature points, the DNA is not being amplified successfully. In this case, give us a call and we will help further troubleshoot with you and help fix your unit!

Q: After staining, I can see the bands from the ladder and the control sample, but not the bands from the experimental samples.

A: If the ladder and control samples are visible, it means that the staining process was successful. This indicates that the issue stems from sample preparation.

• Check that there was no pipetting error. Make sure that students pipetted the correct volumes of the DNA and primer. 
• If the correct volumes were pipetted, make sure that a PCR bead was added to each sample. Also, make sure that the correct type of PCR bead was used. Some kits use the PCR EdvoBeads, while others use the PCR EdvoBeads Plus. 
• If the correct volumes were pipetted and the correct PCR beads were added to each sample, check that the pipettes are calibrated properly. 

Q: After staining, all the DNA bands are very faint.

A: Staining was not successful. Make sure to follow the dilution scheme for SYBR provided in your specific kit, which can be found within the instructor’s guide of the literature. 

• If SYBR Safe DNA Stain was used, make sure that it was diluted properly and the correct volume of diluted SYBR was added to each of your gels. For more information about diluting SYBR, check out this quick guide: A Quick Guide to Using SYBR Safe DNA Stain in EdvoKits
• If SYBR was used, try staining your gel with FlashBlue. If bands are not visible under UV or Blue light, use a FlashBlue staining protocol. Although not the preferred method, it will help visualize those missing bands. 

Q: I spilled my TE buffer, or don’t have enough TE buffer to rehydrate the LyphoControl and/or the LyphoPrimer Mix. What can I replace the TE buffer with?

A: DI water is a great replacement! Use the same volume of TE buffer indicated in the literature with DI water, and rehydrate the LyphoControl/LyphoPrimer Mix.

Q: If my protocol calls for PCR EdvoBeads Plus can I use PCR EdvoBeads instead? Or, vice versa?

A: No, these two types of EdvoBeads are not interchangeable. Please use the type of EdvoBeads that are detailed in your literature.

Q: Why do I need to use/make a control sample? Do I need to add a PCR EdvoBead to a control sample? 

A: A control sample allows you to determine if your thermocycler/cycling program is properly amplifying DNA. 

• If your control sample produces bands on your gel, but your experimental sample does not produce bands, it can help you troubleshoot what went wrong with your experiment.
• The control sample contains all the necessary components for a successful PCR reaction; therefore, a PCR EdvoBead is not needed. Although, make sure to add an EdvoBead to the experimental reactions.

Please reach out to an Edvotek® Technical Support Specialist at 1-800-EDVOTEK or email us at info@edvotek.com if you have any questions.