PCR Clean-up
Contents
1. PCR Clean up User Manual NucleoFast 96 PCR March 2010 Rev 04 MACHEREY NAGEL MN PCR clean up Table of contents 1 Kit contents 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Suitable vacuum manifolds 2 4 Filtration conditions 2 5 Recovery of the purified PCR products 2 6 Automation of NucleoFast 96 PCR 3 Storage conditions 4 Safety instructions 5 General procedure 5 1 Standard protocol for the purification of PCR products vacuum processing 5 2 Standard protocol for the purification of PCR products centrifuge processing 6 Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 Product use restriction warranty ON NODA A Oi 10 10 11 12 14 15 15 16 17 MACHEREY NAGEL 03 2010 Rev 04 PCR clean up 1 Kit contents NucleoFast 96 PCR 4x96 preps 24x 96 preps 10 x 96 preps 50 x 96 preps Cat No 743500 4 743500 24 743100 10 743100 50 esayaly Buffer 50 ml 300 ml i RB RNase free H O 125 ml 2 x 375 ml NucleoFast 96 PCR Plates 4 24 10 ae Elution Plates including Self 4 24 adhering PE Foil User Manual 1 1 1 1 Cat No 743100 10 and 743100 50 do not contain Buffer RB RNase free H O or Elution Plates Composition of Recovery Buffer RB 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 03 2010 Rev 04 PCR clean up 2 Product description 2 1 The basic principle NucleoFast 96 PCR is based on ul2. PCR samples 164 252 359 490 bp upper panel 645 787 982 1484 bp lower panel 25 ul each have been purified manually according to the standard protocol Control unpurified samples Note the high recovery even for small fragments and the efficient removal of primers 2 6 Automation of NucleoFast 96 PCR NucleoFast 96 PCR can easily be automated on common liquid handling instruments As no reassembly of the vacuum chamber is necessary when processing one plate per run NucleoFast 96 PCR can be used fully automated even on workstations without integrated gripper tools During the automated use a recovery volume of 50 ul is recommended Smaller volumes are possible but may lead to a reduced recovery of PCR products and to a lower well to well consistency Recovery can be improved either by mixing incubation or the use of a plate shaker see section 2 5 A very crucial step is the effective recovery of PCR products from the membrane Needles disposable tips have to be as close to the membrane as possible during the recovery step to recover Buffer RB or RNase free H O completely Slight touching of the membrane will not result in damage of the membrane but might block the needles disposable tips during the recovery process resulting in a reduced recovery The height adjustment of the needles disposable tips has to be optimized for each individual platform with extra care for optimal results Make sure that the vacuum is released befo
3. process the NucleoFast 96 PCR Plate If you are not sure that your buckets are able to hold the sandwich of a NucleoFast 96 PCR Plate and a waste collection plate place a standard microtiter plate on top of the appropriate waste collection plate and see if this sandwich fits into the bucket If using a standard Square well Block for waste collection the sandwich hight is 58 mm 1 Transfer the PCR samples 20 100 ul to the NucleoFast 96 PCR Plate Unused wells of the NucleoFast 96 PCR Plate may be left open Sealing is not required 2 Remove contaminants by ultrafiltration Place the NucleoFast 96 PCR Plate onto a suitable waste collection plate e g Square well Block Place the sandwich in the centrifuge and spin at 4 500 x g Note Typically centrifugation for 5 10 min for a sample volume of 50 100 ul is sufficient 3 Washing step Dispense 100 pl RNase free H O into each well of the NucleoFast 96 PCR Plate Place the NucleoFast 96 PCR Plate on top of the waste collection plate and centrifuge for 5 10 min Note The washing step is mandatory if NucleoFast 96 PCR is used under centrifugation About 3 5 ul of PCR sample containing salts primers dNTPs will remain on top of the membrane after the first centrifugation step To avoid contamination of the purified PCR sample the washing step is mandatory to remove the contaminants 4 Recover purified PCR samples Dispense an appropriate volume 25 1
4. 00 ul of Recovery Buffer RB or RNase free H O directly onto the membrane of the NucleoFast 96 PCR Plate Recover DNA by incubation mixing or shaking For more information about the recovery process refer to section 2 5 14 MACHEREY NAGEL 03 2010 Rev 04 PCR clean up 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Insufficient mixing or shaking during recovery step e Increase number of mixing steps increase incubation time optimize shaker speed settings PCR fragment smaller than 150 bp e Use the NucleoSpin 96 Extract II kit for purification of small PCR products Low DNA recovery Recovery buffer volume too small e Increase amount of recovery buffer to at least 25 ul for manual use For automated use a minimum volume of 50 ul is recommended DNA fragments dried onto membrane e Dispense Recovery Buffer RB or RNase free H O and incubate for 15 30 minutes at room temperature to allow DNA to rehydrate before removing DNA Samples not filtered completely e Allow the samples to pass the filter completely Wait until the membrane appears dry and shiny Samples remain on the well s inner wall e Dispense samples directly onto the membrane Make sure Samples are that no sample material sticks to the side of the well as contaminated contaminants might get co recovered Avoid tip touch during automated use of NucleoFast 96 PCR Perform optional washing step No washing step performed while
5. 49 0 24 21 969 270 e mail TECH BIO mn net com Last updated 12 2006 Rev 02 Trademarks Aurum is a registered trademark of Bio Rad Laboratories Inc USA MultiScreen is a trademark of Millipore Corporation USA NucleoFast is a trademark of MACHEREY NAGEL GmbH amp Co KG Vac Man is a trademark of Promega Corporation USA All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we can not grant any guarantees regarding selection efficiency or operation 18 MACHEREY NAGEL 03 2010 Rev 04 Germany and international MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren phone 49 0 24 21 969 0 fax 49 0 24 21 969 199 e mail sales mn net com www mn net com For ordering Germany only Toll free 0800 26 16 000 USA MACHEREY NAGEL Inc 2850 Emrick Boulevard Bethlehem PA 18020 phone 1 484 821 0984 fax 1 484 821 1272 Toll free 888 321 6224 MACH e mail sales us mn net com France MACHEREY NAGEL EURL 1 rue Gutenberg B P 135 F 67722 Hoerdt Cedex phone 33 0 3 88 68 22 68 fax 33 0 3 88 51 76 88 e mail sales fr mn net com Switzerland MACHEREY NAGEL AG Hir
6. AGEL 03 2010 Rev 04 11 NucleoFast 96 PCR vacuum processing 5 1 Standard protocol for the purification of PCR products vacuum processing This protocol is designed for PCR reaction volumes of 20 100 ul For PCR reaction volumes of up to 300 ul filtration times have to be increased The protocol is for manual use or for use with common liquid handling systems 1 Transfer PCR samples to NucleoFast 96 PCR Plate Note Smaller sample volumes should be filled up with H O to 100 ul to enable a uniform loading of the plate Slowly dispense samples directly onto the membrane Avoid dispensing of the samples to the inner wall of the wells Unused wells of the NucleoFast 96 PCR Plate may be left open Sealing is not required 2 Remove contaminants by ultrafiltration Place the NucleoFast 96 PCR Plate on a suitable vacuum manifold and apply vacuum Adjust vacuum to 0 4 to 0 6 bar Note Typically vacuum has to be applied for 10 15 min for a sample volume of 50 100 ul After the samples have passed the NucleoFast 96 PCR Plate completely apply vacuum for an additional 30 60 s 3 Optional washing step Release vacuum 60 90 s Dispense 100 ul RNase free H O into each well of the NucleoFast 96 PCR Plate and apply vacuum 0 4 to 0 6 bar until water has passed the membrane Apply vacuum for an additional 30 60 s Note The optional washing step is recommended if the purity of the PCR samples is
7. ants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict
8. considered not sufficient for desired downstream application If problems after clean up are observed with the downstream application perform the washing step Typically the washing step is not required Reduction of atmospheric pressure 12 MACHEREY NAGEL 03 2010 Rev 04 NucleoFast 96 PCR vacuum processing Recover purified PCR samples Release the vacuum 60 90 s Dispense an appropriate volume 25 100 ul of Recovery Buffer RB or RNase free H O directly onto the membrane of the NucleoFast 96 PCR Plate Recover DNA by incubation mixing or shaking For more information about the recovery process refer to section 2 5 Note Make sure that no vacuum is applied to the manifold when dispensing the recovery buffer MACHEREY NAGEL 03 2010 Rev 04 13 NucleoFast 96 PCR centrifuge processing 5 2 Standard protocol for the purification of PCR products centrifuge processing This protocol is designed for a PCR reaction volume of 20 100 ul For PCR reaction volumes of up to 300 ul filtration times have to be increased This protocol is for manual processing using a microplate centrifuge The centrifuge buckets have to be able to hold the NucleoFast 96 PCR Plate on top of a suitable plate for waste collection e g Square well Block Round well Block not provided with the kit Do not use standard microtiter plates for waste collection as they break under the g forces required to
9. etely from the wells The tips may touch the membrane slightly during the manual recovery process During the automated use a minimum recovery volume of 50 ul is recommended to improve the recovery and the well to well consistency see section 2 6 It is crucial to collect the Recovery Buffer RB completely from the membrane to get an optimal recovery of PCR products The sturdy ultrafiltration membrane allows an easy recovery of purified PCR products without the risk of damaging the membrane Damaging of the membrane would result in the risk of co recovering small membrane parts a common problem with other ultrafiltration membranes These parts might interfere with subsequent applications especially capillary sequencing and microarray spotting With the NucleoFast 96 PCR membrane it is possible to touch the membrane with the tips during the recovery process without the risk of damaging it Recovery of DNA can be facilitated either by a short incubation mixing or by using a plate shaker after the addition of Recovery Buffer RB or RNase free H O this is especially recommended for PCR products 500 bp e Incubate for 5 minutes at room temperature without shaking after the addition of Recovery Buffer RB or RNase free H O MACHEREY NAGEL 03 2010 Rev 04 7 PCR clean up Add Recovery Buffer RB or RNase free H O to the membrane and mix by pipetting up and down 5 10 times or shake for 2 5 minutes on a suitable microplate s
10. haker with moderate shaking For use with a shaker the dispensed recovery buffer volume should be 50 ul When using a plate shaker for recovery the speed settings have to be checked carefully to prevent cross contamination from well to well Proceed as follows Apply 50 100 ul of Recovery Buffer RB or RNase free H O with some added dye e g bromphenol blue to the wells of a NucleoFast 96 PCR Plate Position the plate on the shaker and start shaking with a moderate speed setting for 30 seconds Turn off shaker and check plate surface for small droplets of dyed water Increase speed setting shake for an additional 30 seconds and check plate surface for droplets again Continue increasing the speed setting until you observe droplets on top of the NucleoFast 96 PCR Plate Reduce speed setting check again and use this setting for the recovery step ee paar 0 L 2 a Qa a Qa a a a 2 2 2 a 2 Q 2 a st a Q Q wo N st wo wo DO t co N N o N O t Fragment size Figure 1 The recovery rate depends on the length of the PCR product 100 ul of PCR products have been purified using the NucleoFast 96 PCR Plate under vacuum Mean values and SD of n 8 MACHEREY NAGEL 03 2010 Rev 04 PCR clean up NucleoFast unpurified 96 PCR samples 1 a E primers Figure 2 NucleoFast 96 PCR shows a high recovery even for small fragments
11. liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty MACHEREY NAGEL 03 2010 Rev 04 17 PCR clean up Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel
12. ore MultiScreen Yes Qiagen QlAvac 96 Yes Promega Vac Man 96 vacuum manifold Yes Bio Rad Aurum vacuum manifold Yes Eppendorf Perfect VAC Manifold No 6 MACHEREY NAGEL 03 2010 Rev 04 PCR clean up 2 4 Filtration conditions Filtration time depends on sample volume vacuum strengths and vacuum pump used For use ofthe NucleoFast 96 PCR Plates apply a vacuum of up to 0 6 bar reduction of atmospheric pressure 22 5 inches Hg Use a portable vacuum pump or suitable house vacuum Typically a 100 ul PCR reaction passes the membrane in 10 15 minutes When all of the solution has passed the membrane apply vacuum for an additional 30 60 seconds to allow the liquid to drain off the outlets Before adding Recovery Buffer RB or RNase free H O make sure that vacuum is completely released to prevent the buffer from being sucked through the membrane For processing of the NucleoFast 96 PCR Plates in a centrifuge a force of 4 500 x gis recommended Lower g forces will increase filtration times significantly When using less than 96 samples sealing of unused wells is not required 2 5 Recovery of the purified PCR products Purified PCR products can be recovered directly from the membrane using Recovery Buffer RB or RNase free H O both not supplied with 743100 10 and 743100 50 For manual use the recovery volume should be at least 25 ul Use a multichannel pipettor to recover the buffer containing the purified PCR products compl
13. re recovering the PCR products and adjusting the height of the needles disposable tips as the NucleoFast 96 PCR Plate has a lower position inside the manifold under vacuum This may result in a loss of about 20 30 of PCR fragments If more than one plate is to be processed during the run the plates stored on the platform and currently not in use can be protected with cover lids which are available separately see ordering information MACHEREY NAGEL 03 2010 Rev 04 9 PCR clean up NucleoFast 96 PCR is compatible with common automation workstations Please contact MN or your local distributor for technical support regarding hardware software setup instructions and selection of available protocols 3 Storage conditions All kit components can be stored at room temperature 18 25 C for up to one year 4 Safety instructions All kit components are non hazardous 10 MACHEREY NAGEL 03 2010 Rev 04 NucleoFast 96 PCR 5 General procedure 1 Transfer PCR samples to 20 300 ul NucleoFast 96 PCR Plate 2 Filter contaminants to waste 0 4 to 0 6 bar under vacuum or centrifugation 10 15 min or 4 500xg 10 15 min 3 Wash membrane 100 ul H O RNase free 0 4 to 0 6 bar 10 15 min or 5 10 min 4 500 xg a 4 Recover purified PCR samples 25 100 ul RB or 4 H O RNase free Reduction of atmospheric pressure Optional for vacuum processing MACHEREY N
14. sacker Str 7 P O Box 214 CH 4702 Oensingen phone 41 0 62 388 55 00 fax 41 0 62 388 55 05 e mail sales ch mn net com Service Bioanalysis Technical support and customer service phone 49 0 24 21 969 270 49 0 24 21 969 271 e mail tech bio mn net com
15. trafiltration and designed for rapid clean up of PCR fragments During the procedure the PCR samples are applied to the ultrafiltration membrane Under vacuum or in a centrifuge contaminants primers dNTPs salts are filtered to waste The desired PCR products are retained on the membrane and can be recovered from the membrane after the addition of water or low salt buffer and a short incubation The purified PCR fragments can be used directly for further downstream applications like sequencing or microarray spotting The NucleoFast procedure eliminates the use of chaotropic salts for binding of nucleic acids and subsequent ethanolic washing steps The NucleoFast 96 PCR Plates can be used either manually or automated on standard liquid handling instruments PR dNTP Pine PCR products are loaded directly onto the NucleoFast 96 PCR ie filter membrane PCR products are collected on the surface of the ultrafiltration membrane while contaminants are filtered to waste Optionally 4 the PCR products can be washed with RNase free H O PCR products are recovered from the membrane after addition of water or recovery buffer PCR products are ready to use for downstream applications 2 2 Kit specifications e NucleoFast 96 PCR is designed for the rapid manual clean up of PCR fragments using NucleoVac 96 see ordering information section 6 2 other suitable vacuum manifolds see section 2 3 or microplate centrif
16. uges see section 5 2 Manual processing time for 96 samples is about 20 minutes e NucleoFast 96 PCR can easily be adapted to common liquid handling instruments see section 2 6 The actual processing time for the purification of 96 samples depends on the configuration of the instrument but can be as short as 15 minutes MACHEREY NAGEL 03 2010 Rev 04 5 PCR clean up 2 3 20 300 ul PCR reaction mix can be processed per well If a larger volume is to be processed the sample has to be loaded stepwise Filtration times will increase as the retained PCR products will decrease the permeability of the membrane The recovery volume is gt 25 ul for manual use For automated use a recovery volume 250 ul is recommended High DNA recovery of 50 95 for DNA fragments of 150 bp The purity of recovered PCR products is A JA 1 7 1 8 Purified PCR products are ready to use for downstream applications like automated fluorescent sequencing labelling microarray analysis cloning or restriction digestion The sturdy membrane allows easy recovery of purified PCR fragments without the risk of damaging the membrane No detergents leak out of the membrane Low dead volume of the NucleoFast membrane of 3 4 ul only Suitable vacuum manifolds NucleoFast 96 PCR Plates can be used with common vacuum manifolds Suitable vacuum manifolds Vacuum manifold Suitability NucleoVac 96 Vacuum Manifold Yes Millip
17. using NucleoFast 96 PCR under centrifugation e Perform washing step to remove contaminants MACHEREY NAGEL 03 2010 Rev 04 15 PCR clean up 6 2 Ordering information Product Cat No Pack of NucleoFast 96 PCR Clean up Kit 743500 4 4 x 96 preps 7403500 24 24 x 96 preps NucleoFast 96 PCR Plates 743100 10 10 plates 743100 50 50 plates Cover Lids for NucleoFast 743101 50 50 lids 96 PCR Plates Self adhering PE Foil 740676 50 sheets NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Buffer RB 740362 50 50 ml Square well Block 740670 20 Round well Block 740671 20 Visit www mn net com for more detailed product information 16 MACHEREY NAGEL 03 2010 Rev 04 PCR clean up 6 3 Product use restriction warranty NucleoFast 96 PCR Clean up kit components were developed designed distributed and sold FOR RESEARCH PURPOSES ONLY They are suitable FOR IN VITRO USES ONLY No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoFast 96 PCR Clean up kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warr
Download Pdf Manuals
Related Search