PCR Clean-up for optimal sequencing results. Clean-up results in 5 minutes.
FEATURES
PCR product clean up
5 min protocol
No need for spin columns or magnetic beads
Treatment improves downstream applications such as DNA sequencing and SNP analysis
Ideal for NGS library preparation protocols
DESCRIPTION
PureIT ExoZAP PCR CleanUp Kit consists of a heat labile Exonuclease (HL-ExoI) I and a recombinant Shrimp Alkaline Phosphatase (rSAP).
Treatment of amplified PCR products with PureIT ExoZAP PCR CleanUp Kit, degrades residual primers, single-stranded DNA and inactivates excess dNTPs by dephosphorylation. Add PureIT ExoZAP PCR CleanUp Kit directly to the reaction containing the amplified PCR product. After treatment at 37 °C for minimum 2 minutes both enzymes are completely inactivated by heating at 80 °C for minimum 3 minutes.
PROTOCOL
This protocol serves as a guideline for clean-up of 5 μl PCR product using PureIT ExoZAP PCR CleanUp Kit*.
Take out PureIT ExoZAP PCR CleanUp Kit from the -20 °C freezer.
Keep the components of PureIT ExoZAP PCR CleanUp Kit on ice at all times.
Add 1.5 μl rSAP and 0.5 μl HL-ExoI to 5 μl of amplified PCR product**.
Mix well and spin down.
Incubate the reaction at 37 °C for minimum 2 minutes to degrade remaining primers, single-stranded DNA and to inactivate excess nucleotides by dephosphorylation.
Incubate at 80 °C to completely inactivate both enzymes for minimum 3 minutes.
The cleaned up PCR product can now be used for downstream applications such DNA sequencing, primer extension experiment or SNP analysis.
After treatment the PCR products can be stored at -20 °C
*If treating PCR product of higher volume, then increase proportionally the amount of HL-ExoI and rSAP.
**The enzymes within PureIT ExoZAP PCR CleanUp Kit works in PCR buffers.
PureIT ExoZAP PCR CleanUp IMPROVES QUALITY OF DNA SEQUENCING
PCR product of 866 bp was amplified using TEMPase DNA Polymerase in Ammonium Buffer. The PCR product was spiked with dNTP’s and primers and then treated without and with PureIT ExoZAP PCR CleanUp. Sanger sequencing results of the 866 bp PCR product. The depicted electropherograms are either after treatment with PureIT ExoZAP PCR CleanUp (top) or untreated (bottom). Sequence shown is approximately the region from 250 - 300 bases. Treatment of spiked PCR product with PureIT ExoZAP PCR CleanUp significantly improves the quality of Sanger Sequencing. Treatment enhances signal intensity and eliminates background interference and base miscalls.
