One-step PCR Clean-up for optimal sequencing results. Clean-up results in 5 minutes.
FEATURES
One-step PCR product clean up
Fast 5 min protocol
No Sample Loss
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 consists of a balanced combination 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, removes residual primers, single-stranded DNA and inactivates excess dNTPs by dephosphorylation. Add PureIT ExoZAP PCR CleanUp directly to the reaction containing the amplified PCR product. After treatment at 37 °C for minimum 2 minutes, PureIT ExoZAP PCR CleanUp is 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*.
Take out PureIT ExoZAP PCR CleanUp from the -20 °C freezer.
Keep PureIT ExoZAP PCR CleanUp on ice at all times.
Add 2 μl PureIT ExoZAP PCR CleanUp** to 5 μl of amplified PCR product.
Mix well and spin down.
Incubate the reaction at 37 °C for 2 - 5 minutes to degrade remaining primers, single-stranded DNA and to inactivate excess nucleotides by dephosphorylation.
Incubate at 80 °C to completely inactivate PureIT ExoZAP PCR CleanUp for 3 - 10 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 PureIT ExoZAP PCR CleanUp.
**PureIT ExoZAP PCR CleanUp 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 (A) without PureIT ExoZAP PCR CleanUp (B) with PureIT ExoZAP PCR CleanUp (2 +3 min) and (C) with an enzymatic PCR clean-up reagent from a competting brand (1+ 4 min) prior to Sanger sequencing. All samples were analyzed in triplicates and according to manufactures recommendations. Sanger sequencing result was compared by graphically plotting the number of base calls with certain quality values (Phred score).
Treatment of PCR products with PureIT ExoZAP significantly improves the quality of DNA sequencing. Furthermore, PureIT ExoZAP PCR CleanUp showed PCR clean-up results equivalent to the competing PCR clean-up reagent.
*Data with Quality values less than 10 is not included.
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.
No sample loss after treatment with PureIT ExoZAP PCR CleanUp.
Human gDNA were amplified using primers targeting four different DNA targets. GADPH 125 bp (lane 1-2), DMD17 400 bp (lane 3-4), CSTR3053 3053 bp (lane 5-6) and BRCA2.8 5575 bp (lane 7-8). M is Ampliqon High Range DNA Ladder. PCR products were loaded onto 1 % DNA agarose gel after treatment with Buffer P (-) or with (+) PureIT ExoZAP PCR CleanUp. It is clearly observed that PCR products of variant target lenghts can be treated with PureIT ExoZAP PCR CleanUp with no sample loss.
Buffer P has same buffer composition as PureIT ExoZAP PCR CleanUp, but without HL-ExoI and rSAP.
