SequalPrep™ Long PCR Kit with dNTPs - FAQs

View additional product information for SequalPrep™ Long PCR Kit with dNTPs - FAQs (A10498)

13 product FAQs found

What is the fidelity of the SequalPrep Long Polymerase in SequalPrep Long PCR Kit with dNTPs?

The SequalPrep Long PCR Kit with dNTPs demonstrates fidelity 3-4 times greater than recombinant Taq polymerase, similar to Invitrogen’s Platinum Taq High Fidelity Enzyme (by rpsl assay).  The SequalPrep Long PCR Polymerase was compared to rTaq, Platinum Taq HiFi, & Roche's Expand Long Template PCR System.  Relative fidelity:
• rTaq = 1
• Platinum Taq HiFi = 3.3
• Roche = 3.6
• SequalPrep Long with Enhancer A = 3.6
• SequalPrep Long with Enhancer B = 4.4

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What organisms have been tested with SequalPrep Long PCR Kit with dNTPs kit?

Human and mouse genomic DNA have both been tested with the kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

SequalPrep Long PCR Kit with dNTPs comes with several enhancers. What are they and when should I use them?

The two Enhancers were selected to help provide maximum success rates. We recommend setting up the initial reactions with 0.5X Enhancer A to provide maximum success rate.  For any unsuccessful amplicons we recommend testing the following conditions to optimize amplification:
• 1.0X Enhancer A
• 0.5X Enhancer B
• 1.0X Enhancer B

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

How many units of the SequalPrep Long Polymerase should I use in a typical reaction for SequalPrep Long PCR Kit with dNTPs?

We recommend using 1.8 Units of SequalPrep Long Polymerase in a reaction.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What amount of input material do you recommend for SequalPrep Long PCR Kit with dNTPs?

We recommend 5-200 ng of human genomic DNA and 20-80 ng of mouse embryonic stem cells DNA. 50 ng of DNA template at a concentration of 50 ng/µl should be sufficient for most applications, although input amounts as low as 10 ng can be possible depending on the sample type, due to the high yields generated by the kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What reaction volume should I use for SequalPrep Long PCR Kit with dNTPs?

We recommend setting up a 20 µL reaction.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is the largest DNA fragment that I can amplify with SequalPrep Long PCR Kit with dNTPs?

The SequalPrep Long PCR Kit with dNTPs has been used to amplify amplicons from 5-20 kb with a very high success rate. Depending on reaction conditions (template prep, primer design, enhancer selected, etc.) templates have been amplified up to 22 kb. We recommend that you determine the optimal condition for successful amplification of your template.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What enzyme is used in SequalPrep Long PCR Kit with dNTPs?

The SequalPrep Long PCR Kit with dNTPs uses the SequalPrep Long Polymerase. This polymerase was selected to provide maximum length, fidelity, and maximum success rate for long amplicons. Hot-start functionality is also included to increase specificity of the kit.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Can I use the SequalPrep Long PCR Kit with dNTPs for applications outside of Next Generation Sequencing sample enrichment?

Yes, we recommend using the SequalPrep Long PCR Kit with dNTPs for performing long range PCR for any application.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Does the SequalPrep Long PCR Kit with dNTPs replace reagents currently in the Next Generation Sequencing Process workflow?

No, the SequalPrep Long PCR Kit with dNTPs is used to enrich the genome for areas of interest before using any of the recommended kits for the NGS workflow (tagging, fragmentation, etc).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

What is causing PCR amplification in my negative controls when I use SequalPrep Long PCR Kit with dNTPs?

Amplification of your PCR product in negative controls is caused by contamination. We recommend the following to resolve the issue: - Replace all of your reagents, including the water. - Use aerosol-resistant pipette tips. - Use separate workstations for PCR set-up and PCR product analysis.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

I used the SequalPrep Long PCR Kit with dNTPs and when I run the product on a gel I get a smear or non-specific bands. Do you have any tips for troubleshooting?

There are multiple factors that can result in PCR products that produce a smear of DNA or non-specific bands on an electrophoresis gel. Potential causes as well as recommendations for troubleshooting each are below:
1. Low annealing temperature - primers can bind off-target sequence if the annealing temperature is too low. To improve specific binding we recommend that you:
  • Increase the primer annealing temperature, up to a maximum of 68°C.
  • Set up a gradient PCR to determine the optimal annealing temperature for your primer pairs.
2. Sub-optimal cycling parameters - we recommend optimizing your PCR cycling conditions by trying the following:
  • Reduce the number of cycles in your PCR program.
  • Reduce the length of one or more of your cycle segments (denaturation, annealing, or extension).
3. Primers - issues with incorrect primer storage, poor primer quality, as well as sub-optimal primer design or concentration can negatively affect PCR amplification. To address primer issues we recommend the following:
  • Make aliquots of the primer stock and store primers at -20°C.
  • Make sure primers are not degraded.
  • If working with established primer pairs, check primer performance with a control template, under established PCR conditions.
  • Review primer design and if needed, design alternative primer pairs.
  • Determine the optimal primer concentration for the reaction. The final concentration range is generally 0.3-0.6 µM.
  • Make sure sense and anti-sense primers are present in equal concentrations.
  • For targets ≥15 kb, design multiple primer pairs to increase the chance of amplification.
4. DNA template is degraded or at the wrong concentration - degraded DNA template, as well as adding too much or too little of the DNA template can result in poor yield from your PCR. To troubleshoot this issue:
  • Test serial dilutions of your DNA template.
  • Check the quality and purity of your DNA.
5. Contamination with previous PCR product or other non-target template - to resolve poor amplification caused by contamination, we recommend that you:
  • Replace all of your reagents.
  • Use separate areas for PCR set-up and PCR product analysis.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

I used the SequalPrep Long PCR Kit with dNTPs and my reaction failed to amplify. How can I troubleshoot the reaction?

Poor amplification of the PCR reaction can be due to different issues. Please see our recommendations below depending on the possible causes:
1. Poor DNA quality - to help enusre good quality DNA for PCR we recommend that you:
  • Check the quality and concentration of your template.
  • Include a positive control reaction using a known template under established PCR conditions.
  • Check your template purification process. Make sure there are no PCR inhibitors present such as ionic detergents, heparin, phenol, xylene cyanol, bromophenol blue, etc. You can test for presence of inhibitors in the template by spiking the original PCR mix with dilutions of a template that you know amplifies.
  • Analyze the template on an agarose gel to ensure no degradation.
2. Difficult templates (GC-rich or high secondary structure) - to address PCR issues due to difficult templates we recommend that you:
  • Lengthen the initial denaturation step, increase the denaturation temperature (98°C maximum), increase the annealing temperature (68°C maximum), or combine 2-3 of these modifications.
  • Titrate DMSO concentration from 2-10% of the final reaction.
  • Reduce the elongation temperature down to 58°C for low-GC content templates.
3. Sub-optimal primer design or concentration - to address PCR amplification issues caused by primer design and concentration we recommend that you:
  • Titrate primer concentration to a final concentration of 0.3-0.6 µM in the reaction.
  • Design alternative primers that meet the following parameters:
  • 40-60% GC content
  • 3' GC-clamp (at least 1 or 2 Gs or Cs)
  • Similar primer Tm values, size, and nucleotide ratios
  • Avoid repetitive motifs, hairpins, palindromes, and long stretches of polypurines and/or polypyrimidines
4. Sub-optimal cycling conditions: to address poor amplification caused by cycling conditions we recommend that you:
  • Reduce the annealing temperature.
  • Increase the number of cycles.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.