eLabNotebook - Millipore's Fully Automated Method for PCR Purification Using Montage* PCR96 Cleanup Kits on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > PCR Cleanup > Millipore Montáge* PCR₉₆
Cleanup Biomek® FX

Millipore's Fully Automated Method for PCR Purification Using Montáge* PCR₉₆ Cleanup Kits on the Biomek® FX Nucleic Acid Preparation System

Millipore– www.millipore.com

Configuration of the Biomek FX® Worksurface

(Click to Enlarge)

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:	MultiScreen96-PCR? plate
P10:	Reservoir for tip washing
P11:	Reservoir w/resuspension solution
P12:	Thermocycler plate with PCR samples

Procedure

	1.	Transfer 100µl 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 100µl 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 100µl PCR reaction. Samples were filtered through a MultiScreen96-PCR plate for 10 minutes and recovered in 100µl of resuspension solution by mixing. M=Low DNA Mass ladder (8µl, Gibco Life Technologies). 10µl of purified PCR samples were loaded into each well of a 1% agarose gel. U=Unpurified PCR reaction (10µl).

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. 100µl PCR reactions (657bp) were purified on the MultiScreen96-PCR plates from the Millipore Montage PCR Cleanup Kit. Samples were resuspended in 100µl 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.

* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.