Skip to Store Area:

Sciresys LLC
  • Toll Free 888-666-0536

You're currently on:

Search Site
 

96-well PCR Product Purification

96-well PCR Product Purification Kit：Purification of 96 PCR products within 30 min

Contents

Introduction

The EZgene™ families of products are innovative systems that radically simplify extraction and purification of nucleic acids from a variety of sources. Key to the system is the new matrix that specifically, but reversibly, binds DNA or RNA under certain optimal conditions, allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or low salt buffer. The 96-well Cycle Pure Kit is a convenient system for fast and reliable purification of up to 96 PCR products. The method uses ezBind technology to recover DNA bands 50 bp-40 kb free of oligo nucleotides, nucleotides, and polymerase in yields exceeding 80%. Binding conditions are adjusted by addition of a specially formulated buffer, and the sample is applied to a DNA Plate. Following a rapid wash step, DNA is eluted with deionized water (or low salt buffer) and ready for other applications. No organic extractions or alcohol precipitations means safe and rapid processing of multiple samples in parallel. The product is suitable for T-A ligations, PCR sequencing, restriction digestion, or various labeling reactions. In addition the kit can be used to purify DNA from any other enzymatic reaction.

Benefits Cycle-Pure Kit means:

R Speed: Up to 96 DNA products can be recovery from enzymatic reactions <25 min

R Reliability: Optimized buffers guarantee pure DNA

R Safety: No organic extractions

R Quality: Purified DNA suitable for any application

2 Item(s) Show per page
View as: Grid  List  Sort by: Best Value| Name| Price
96-well Cycle pure kit
Cat#: DC3812-01
Size: 4x96
Purification of 96 PCR products within 30 min Learn More
$ 440.00
Add to Cart
96-well Cycle pure kit
Cat#: DC3812-02
Size: 10x96
Purification of 96 PCR products within 30 min. Learn More
$ 2,000.00
Add to Cart
   
2 Item(s) Show per page
View as: Grid  List  Sort by: Best Value| Name| Price

Storage and Stability All 96-well Cycle Pure Kit components are guaranteed for at least 24 months from the date of purchase when stored at 22-25℃. Under cool ambient conditions crystals may form in Buffer GC. Simply warm at 37℃ to dissolve.

Binding Capacity Each well on the 96-well DNA Plate can bind 1~12 μg DNA.

Kit contents:

Catalog#

DC3812-00

DC3812-01

DC3812-02

Purification times

1 x 96

4 x 96

20 x 96

96-well™ DNA Plates

1

4

20

Racked Microtubes (1.2 mL)

1

4

20

8-Strip Microtube Caps

24 x 8

50 x 8

288 x 8

96-well Collection Plates (2 mL)*

1

2

4

Buffer GC

60 mL

2 x 160 mL

3 x 480 mL

DNA Wash Buffer

40 mL

100 mL

2 x 200 mL

Elution Buffer

15 mL

60 mL

300 mL

User Menu

1

1

1

2 mL collection plate can be cleaned and reused. See page 7 for details. Before Starting Please read this booklet thoroughly to ensure that you are familiar with the entire procedure. 96-well Cycle Pure Kit is designed to be simple, fast, and reliable provided that all steps are followed diligently. All steps must be performed at room temperature.

IMPORTANT

Dilute DNA Wash Buffer with absolute ethanol as follows: DC3812-00:Add 160 mL absolute ethanol to each bottle DC3812-02:Add 400 mL absolute ethanol to each bottle DC3812-03:Add 800 mL absolute ethanol to each bottle

Store the diluted DNA Wash Buffer at room temperature!

96-well Cycle Pure Vacuum Manifold Protocol Materials Supplied By User

R Vacuum manifold which can fit the 96-well plate and the 96-well collection plate. (For vacuum protocol).

R Centrifuge with swinging rotor which is capable of 4000 x g (such as Eppendof 5810 with MTP rotor or Beckman Allegra 6 with PTS-2000 rotor.) (For centrifugation protocol).

R Protective eye-ware.

R Absolute (96% - 100%) ethanol.

R Necessary accessories such as plate seals and lids.

R Protective eye-ware.

　

1. Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.

2. Determine the volume of the PCR reaction. Add 2 volumes of Buffer GC to 1 volume of PCR sample and mix. For PCR products <200 bp add 3 volumes of Buffer GC. Vortex thoroughly to mix.

Prepare the vacuum manifold according to the instruction of the manufacturer. For Vac-3 manifold, place a 2 mL collection plate inside the manifold and place the top plate of manifold squarely over the base. Apply the samples to the wells of the 96-well™ DNA Plate. Seal the unused wells with sealing film tape and place the 96-well™ DNA Plate over the top plat of the manifold.

3. Turn on the vacuum manifold and filter through the mixtures by vacuum.

4. Discard the pass through liquid.

5. Wash the plate by adding 800 μL of DNA Wash Buffer diluted with absolute ethanol. Vacuum through for 5 min at room temperature. Discard liquid, reuse the collection plate and repeat step 5 with another 800 μL of DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with absolute ethanol before

use. Refer to label on bottle for directions.

6. Discard liquid and vacuum 10 more min to dry the resin.

7. Remove the 96-well™ DNA Plate from the manifold, gently tap the plate on a stack of absorbent paper until no liquid drops come out. This step will ensure the removal of residual DNA Wash Buffer from outlet of nozzles of the 96-well™ DNA Plate. Residual of ethanol, which is in DNA Wash Buffer, may interfere downstream enzymatic reactions.

8. Optional: Place the 96-well™ DNA Plate into a vacuum oven preset at 70℃ for 10 min to further dry the plate. (This step will ensure that the DNA plate is completely dried before elution.).

9. Assemble the manifold by placing the racked microtubes inside the based of manifold. Place the 96-well™ DNA Plate on top part of the manifold.

10. Place 96-well™ DNA Plate on top of the vacuum manifold. Add 80-100 μL (depending on desired concentration of final product) Elution Buffer (10 mM Tris, pH 8.5 ) directly onto the resin in each well and turn on the vacuum for 5 min to elute DNA. This represents approximately 80-90% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

96-well Cycle Pure Spin Protocol

1. Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.

2. Determine the volume of the PCR reaction. Add 2 volumes of Buffer GC to 1 volume of PCR sample and mix. For PCR products <200 bp add 3 volumes of Buffer GC. Vortex thoroughly to mix.

3. Place the 96-well™ DNA Plate on top of the 2mL collection plate and transfer samples to the 96-well™ DNA Plate. Put them into a microplate rotor.

4. Centrifuge at 3000-4000 x g for 5 min.

5. Discard the flow-through by invert the 2 mL collection plate to a waste container. Reuse the collection plate.

6. Add 800 μL DNA wash buffer to each well of the 96-well™ DNA Plate. Centrifuge at 3000-4000 x g for 5 min. Repeat this step with another 800 uL DNA wash buffer. Centrifuge at 4000 x g for 10 min.

Note: DNA Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.

7. Optional: Remove the DNA plate and place it into a vacuum oven or incubator which was preset to 70℃ for 10 min. This step will ensure that the DNA plate is completely dried before DNA elution.

8. Add 80-100 μL of Elution Buffer (10 mM Tris, pH 8.5) to each well of the DNA plate.

9. Carefully place the 96-well™ DNA plate on top of the racked microtubes® Centrifuge at 4000 x g for 5 min to elute DNA. This represents approximately 75%-80% of bound DNA. An optional second elution will yield any residual DNA, though at lower concentration.

10. Yield and quality of DNA: determine the absorbance of an appropriate

dilution of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows:

DNA concentration=Absorbance260 × 50 × (Dilution Factor) μg/mL Fragments greater than 500 bp in length can routinely be purified at >80% yield. Bands ranging from 50 bp to 500 bp give yields of 60%-90%. The ratio of (A 260)/ (A280) is an indication of nucleic acid purity. A value greater than 1.8 indicates > 90% nucleic acid. Alternatively, yield (as well as quality) can sometimes best be determined by agarose gel/ethidium bromide electrophoresis. Cleaning of 2 mL 96-well Plates The 2 mL 96-well collection plates are reusable. To avoid cross-contamination, rinse the plates throughly with tap water after each user. Rinse with 0.5 M HCl for 5 min and water throughly with distilled water. 2 mL 96-well collection plates can also be autoclaved after wash. Short Protocol For Experienced Users (Vacuum Protocol)

1

Determine volume of reaction. Add 2 volumes of Buffer GC to PCR reaction.

2

Apply solution to 96-well DNA plate assembled in vacuum manifold.

3

Turn on the vacuum and filter through the mixtures by vacuum.

4

Wash plate with 800 μL each well by vacuum suction.( DNA Wash Buffer should be diluted with ethanol before use).

5

Dry the membrane by 10 min further vacuum.

6

Place column into clean 96-well collection plate and elute DNA with 80-100 μL sterile water or TE Buffer by vacuum suction.

Trouble Shooting Guide

Problem

Possible reason

Suggested improvement

Low DNA yields

Too little Buffer GC added to sample

Add more Buffer GC as indicated. For DNA fragments <200 bp in size, add up to 6 x vol Buffer GC. For DNA fragments > 4 kb, add 3 volumes of Buffer GC followed by 1 volume distilled water.

pH of water for elution is<7.5

Check the pH of the water, adjust the pH of the water to 8.0 using Tris-HCl.

No DNA eluted.

DNA Wash Buffer Concentrate not diluted with absolute ethanol

Prepare DNA Wash Buffer Concentrate as instructed above.

Optical densities Do not agree with DNA yield on agarose gel

Trace contaminants eluted from column increase A260.

Make sure to wash column as instructed in steps 4 and 5. Alternatively, rely on agarose gel/ethidium bromide electrophoresis for quantization.

DNA sample floats out of well while loading agarose gel

Ethanol not completely removed from column following wash steps.

Vacuum or centrifuge the plate as instructed and incubate the plate before proceeding to elution step.