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Fully Automated Method for PCR Purification Using Montage™ PCR96 Cleanup Kits on the Biomek® FX Nucleic Acid Preparation System		Tech Note - TN1105EN00 >  Product Catalogue

Here we describe a fully automated PCR purification method using Millipore’s Montage PCR₉₆ Cleanup Kit on the Beckman Biomek FX pipettor. The procedure requires no manual intervention after the deck is set up. The process was set up on an instrument with one 96 channel pipetting head and one vacuum manifold on the deck.

To increase the throughput of this process, the deck can be set up with four vacuum manifolds to handle multiple plates simultaneously. Purification conditions may be optimized for individual needs. The protocol includes one filtration step followed by resuspension and recovery of the sample in a volume to be specified by the user. The procedure yields one plate of 96 purified products in less than 15 minutes.

Configuration of the Biomek FX Worksurface

Prior to starting:
Make sure deck is set up as follows:

SPE1: Assembled Millipore manifold
P7: P200 Tips
P8: V-bottom collection plate for storage
P9: MultiScreen®₉₆-PCR plate
P10: Reservoir for tip washing
P11: Reservoir w/resuspension solution
P12: Thermocycler plate with PCR samples

Procedure

1. Transfer 100 µL of unpurified PCR product from thermocycler sample plate (P12) to MultiScreen₉₆-PCR plate (P9). Wash tips before unloading.

2. Move MultiScreen₉₆-PCR to Vacuum Manifold at SPE1.

3. Apply vacuum for 10 minutes.

4. Move MultiScreen₉₆-PCR back to position P9.

5. Load tips to add 100 µL resuspension solution from reservoir on P11 to MultiScreen₉₆-PCR plate (P9).

6. Mix each sample 25 times at 100% speed and recover to collection plate (P8).

Figure 1. Agarose gel of purified PCR products

Figure 1. Representative samples from PCR purification using a Millipore Montage PCR₉₆ Cleanup Kit on the Beckman Biomek FX. A 657 base pair product was amplified in a 100 µL PCR reaction. Samples were filtered through a MultiScreen₉₆-PCR plate for 10 minutes and recovered in 100 µL of resuspension solution by mixing. M=Low DNA Mass ladder (8 µL, Gibco Life Technologies). 10 µL of purified PCR samples were loaded into each well of a 1% agarose gel. U=Unpurified PCR reaction (10 µL).

Table 1. Average product recovery (%)

657 base pair product
Mean	110%
Stdev	21%
CV	19%
Min	66%
Max	136%

Table 1. Recovery of purified PCR products seen in Figure 1. 100 µL PCR reactions (657bp) were purified on the MultiScreen₉₆-PCR plates from the Millipore Montage PCR Cleanup Kit. Samples were resuspended in 100 µL of TE buffer. Percent recovery was determined by gel electrophoresis, ethidium bromide staining and densitometry. Error rates for this analysis method can be 10–15%.

Conclusion:

Using the Millipore Montage PCR₉₆ Cleanup Kit allows for removal of excess primers and salts from the starting PCR reaction mix. Recoveries for a 657bp PCR product are 90% or greater for the conditions outlined in this procedure. The purified products may be used for downstream applications such as sequencing, microarrays and restriction digest without further purification. The process is simple, fast and automation compatible.

Ordering Information

Product Name	Qty./Pk	Catalogue Number
PCR96 Cleanup Kit	1 x 96-well plate	LSKC09601
PCR96 Cleanup Kit	4 x 96-well plate	LSKC09604
PCR96 Cleanup Kit	24 x 96-well plate	LSKC09624

Product Name	Catalogue Number
Millipore Vacuum Manifold	MAVM0960R
Beckman Assembly Kit	MAVMMEK20

Millipore and MultiScreen are registered trademarks of Millipore Corporation.

Montage is a trademark of Millipore Corporation.
Biomek is a registered trademark of Beckman Instruments, Inc.

Copyright © 2001 Millipore Corporation. All rights reserved.