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AuPreP™ PCR Purification Kit (50 preps)


Cat.No. PP28-104LT
Cat.No. PP28-106LT


Description
Purification of small-scale DNA using phenol/chloroform extraction and ethanol precipitation is laborious and time-consuming. AuPreP™ PCRP Purification Kit provides a simple and fast method to purify and clean up PCR products or DNA fragments from components of enzymatic reactions from enzymes, dNTPs, salts and primers without phenol/chloroform extraction. It is based on the phenomenon of binding of up to 20 µg DNA to silica-based membranes in chaotropic salts with average recoveries from 60 to 95% of 100-bp to 10-kb DNA fragments.
Parameter Value
Average preparation time

5 ~ 10 minutes
Workable length of fragment 100-bp ~ 10-kb
Maximal recovery ~95%
Minimal capacity Up to 20 μg
Regular sample volume 10 ~ 100 mg
 

Downstream Applications
  • Restriction enzyme digestion
  • Modifying enzymatic reaction
  • Radioactive and Fluorescent labeling & sequencing
  • Sequencing & PCR
  • Ligation
  • Labeling & Hybridization

Product Contents
 
  PP28-104LT (50Preps.) PP28-106LT(250Preps.)
PX Buffer

30ml

 150ml
WN Buffer 6ml* 30 ml**
WS Buffer 6 ml+ 30 ml
Elution Buffer 5ml 25 ml++
PCRP Column 50 pieces 250 pieces
Collection Tube 50 pieces 250 pieces
Protocol 1 1
 
For GX28-704LT
Add 24 ml of 98-100% ethanol into WN and WS Buffer before first use. Please be sure to tighten the cap after each use when the ethanol has been added.
For GX28-706LT
Add 120 ml of 98-100% ethanol into WN and WS Buffer before first use. Please be sure to tighten the cap after each use when the ethanol has been added.
Shipping and Storage
AuPreP™ PCRPPurification Kit is stable at 20-250C for one year. Product should be stored in dry place and kept away from direct sunlight.
Important Notes

Please read the following notes before starting the procedures.
  • Buffers provided in this Kit contain irritants. Appropriate safety apparels such as gloves and lab coat should be worn.
  • All procedures should be done at room temperature (20-250C).
  • For 50 preps, add 60 ml of 98-100% ethanol into WS Buffer bottle when first open. For 250 preps, add 180 ml of 98-100% ethanol into WS Buffer bottle when first open. Ethanol is provided by the user.
  • All centrifugation steps are done at full speed (10,000 x g or 13,000-14,000 rpm) in a microcentrifuge.
  • DNA can be eluted in Elution Buffer (provided), Milli-Q or double-distilled H2O , or TE buffer. Since DNA elution only takes place effectively at pH 7.0 - 8.5, make sure that the pH of H2O is within this range (refer to AuPreP™ PCRP Hints, No. 1, page 14). For long-term storage of the eluted DNA, TE buffer should be used for elution. Since EDTA in TE may affect downstream applications, Elution Buffer or H2O is preferred for elution of DNA immediately used for further enzymatic
 

AuPreP™ PCRP PURIFICATION KIT----------------- Protocol for Spin Method
 
  Related Notes
1. Pipet 10-100µl PCR* product (make sure that mineral oil is not taken) or DNA solution after enzymatic reaction to a new 1.5-ml centrifuge tube. Add 0.5 ml PX Buffer and mix well.

If more than 100μl is used in order to harvest more DNA, increase the applied volume of PX buffer proportionally.
2. Place a PCRP Column onto a Collection Tube. Add all the mixture from step 1 into the column. If the volume of mixture is more than 0.7 ml, load and filter 0.7 ml at each time until all the mixture has been filtrated.
3. Centrifuge for 30-60 seconds. Discard the flowthrough.  
4. Wash the column once with 0.5 ml WN Buffer by centrifuging for 30-60 seconds. Discard the flowthrough. Ensure that the ethanol has been added into WN and WS buffer before first use.
5. Wash the column once with 0.5 ml WS Buffer by centrifuging for 30-60 seconds. Discard the flowthrough. Keep the cap of the WN and WS bottle tight after each use to avoid volatilization of ethanol. Decreased ethanol content in WN or WS buffer may cause DNA loss during the wash.
6. Centrifuge the column at full speed for another 3 minutes to remove residual ethanol. Residual ethanol can affect the quality of DNA and inhibit subsequent enzymatic reactions or sequencing. If necessary, stand the column at room temperature for a few minutes before eluting DNA.
Do NOT remove ethanol by baking the column into an oven, as high temperature may affect the intactness of the column.
7. Place the column onto a new 1.5-ml centrifuge tube. Add 15-30µl of Elution Buffer (provided) onto the center of the membrane. Use of elution solution preheated to 60ºC can increase the recovery of DNA fragment larger than 5-kb.

For effective elution, make sure that the elution solution is dispensed onto the center of the membrane and is completely absorbed (refer to AuPreP™ PCRP Hints , No. 2 & 3, page 14).

If the solution still retains on the surface, pulse-centrifuging the tube for 1-2 seconds can drag the solution into the membrane. Do NOT over-centrifuge as the solution will get out of the membrane easily.
8. Stand the column for 3 minutes, and centrifuge for 1- 2 minutes to elute DNA. Higher DNA recovery can be attained by eluting the column twice. That is, elution twice with, e.g. 30 μl H2O or buffer, yields more DNA in total than eluting once with 60 μl H2O or buffer.
9. Store DNA at –20ºC.  
 

AuPreP™PCRP PURIFICATION KIT --------- Protocol for Vacuum Method
 
  Related Notes
1. Pipet 10-100µl PCR* product (make sure that mineral oil is not taken) or DNA solution after enzymatic reaction to a new 1.5-ml centrifuge tube. Add 0.5 ml PX Buffer and mix well.

Make sure that NO MINERAL OIL is taken. Any mineral oil will adversely interfere with the following protocol.
2. Insert a PCRP Column into the luer-lock of a vacuum manifold (e.g. Promega's Vac-man**). Add all the mixture from step 1 into the column Load no more than 0.7 ml mixture into the column each time until all the mixture has been filtrated.
3. Apply vacuum to draw all the liquid into the manifold.  
4. Wash the column once with 0.5 ml WN Buffer by reapplying vacuum to draw all the liquid. Ensure that ethanol has been added into WN & WS Buffer bottle when first use.
5. Wash the column once with 0.5 ml WS Buffer by reapplying vacuum to draw all the liquid. Keep the cap of the WN and WS bottle tight after each use to avoid volatilization of ethanol. Decreased ethanol content in WN or WS buffer may cause DNA loss during the wash.
6. Place the column onto a Collection Tube. Centrifuge the column at full speed for 3 minutes to remove residual ethanol. Residual ethanol can affect the quality of DNA and inhibit subsequent enzymatic reactions. If necessary, centrifuging the column for a few minutes more can remove all the ethanol before eluting DNA. However, do NOT remove ethanol by putting the column into an oven as high temperature may affect the intactness of the column.
7. Place the column onto a new 1.5-ml centrifuge tube. Add 15-30µl of Elution Buffer (provided) onto the center of the membrane. Use of elution solution preheated to 60ºC can increase the recovery of DNA fragment larger than 5-kb.

For effective elution, make sure that the elution solution is dispensed onto the center of the membrane and is completely absorbed (refer to AuPreP™ PCRP Hints , No. 2 & 3, page 14).
8. Stand the column for 1-2 minutes, and centrifuge for 1-2 minutes to elute DNA Higher DNA recovery can be attained by eluting the column twice. That is, elution twice with, e.g. 30 μl H2O or buffer, yields more DNA in total than eluting once with 60 μl H2O or buffer.
9. Store DNA at -20ºC.  
* PCR is covered by U.S. patents 4,683,195 and 4,683,202 issued to Hoffmann-La Roche Inc.
** Vac-man is a trademark of Promega Corporation.
 

Troubleshooting Guide
 
Problem Possible Reason Solution
Low recovery of DNA fragment

DNA solution used is more than 100µl

 Divide loading the sample into two or more columns.

If DNA to be cleaned up is diluted, more than 100 µl solution can be used per column. Add 5µl of more PX Buffer for each 1 µl of extra DNA solution (e.g. add 600 µl PX Buffer to 120µl DNA solution).
  Ineffective DNA elution DNA elution does not take place well at acidic conditions. Make sure that water or buffer is of pH between 7.0 and 8.5
  Size of DNA product is more than 5-kb Use elution solution preheated to 60ºC.
  Eluted DNA carries salt residue Wash the column twice with 0.5ml WS buffer.
  Incomplete DNA elution Complete DNA elution only takes place when elution solution is in full contact with the membrane. Make sure that no less than 15 µl of solution is dispensed onto the membrane and is completely absorbed into it before centrifugation.
Poor performance in downstream applications Eluted DNA carries ethanol residue After wash with WS Buffer, do discard the flow-through, and centrifuge the column for another 3 minutes. If necessary, centrifugation for a few minutes more can completely remove ethanol. However, do not remove ethanol by putting the column into an oven as high temperature may affect the intactness of the column.
Poor OD260/OD280 ratio Use of H2O of acidic pH to dilute the eluted DNA Make sure the H2O has the pH value between 7.0-8.5
 

AuPreP™ PCRP Hints
 
  • Milli-Q or double-distilled H2O stored in a laboratory for a period of time usually becomes acidic due to dissolution of CO2 or other acidic vapor such as HCl from air. Always check the pH to make sure that it is between 7.0 to 8.5 before used. Use H2O of pH less 7.0 for elution will lead to reduced yield of DNA. Use H2O of acidic pH (pH 5.0-6.0) to dilute DNA or RNA samples for spectrophotometric analysis will also significantly decrease A260/A280 ratio of the sample (Wilfinger et al., 1997).

  • Higher DNA recovery can be attained by eluting the column twice. That is, eluting twice, e.g., with 30 µl H2O or buffer, yields more DNA in total than eluting once with 60 µl H2O or buffer.

  • Use of elution solution preheated to 60°C can increase recovery of DNA fragment larger than 5 kb.
 

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