96-Well Plate Blood Genomic DNA Mini-Preps Kit

KitContents:

Note 1: Universal Buffer CL containschaotropic salt. Avoid contact with skin and eyes.

Note 2: CW1 Solution and CW2 Solution aresupplied as concentrates. Add 68ml ofethanol (96-100%) to 52ml of CW1 Solution; add 85ml ofethanol (96-100%) to 65 ml of CW1 Solution, add 84ml ofethanol (96-100%) to 36ml of CW2 Solution; add 105ml ofethanol (96-100%) to 45ml of CW2 Solution before use to obtain working solutions.

Storage and Stability

96 Well Plates and all buffers should be stored dry, at roomtemperature (15-25°C)and are stable for 1 year under proper storage. Proteinase K is supplied as 10mg/ml solution, the solution can be keep at 4°C for 6 months, for long-termstorage keep at -20°C.

Introduction

96-Well Plate blood genomic DNA minipreps kit is designedfor rapid and high-throughout purification of genomic DNA from fresh or frozenanticoagulated blood.

Samples are first lysed using proteinase K in an optimizedbuffer. The lysate is then loaded onto the 96 well BP plate, DNA is selectivelybound to the BP membrane embed in the plate in the presence of highconcentrations of chaotropic salt. During wash steps, protein and otherimpurities are removed and DNA is then eluted in water or low-salt buffer.Purified DNA typically has an A₂₆₀/A₂₈₀ ratios of1.7-1.9, and is highly suited for most downstream applications such as PCR,Southern blotting, RAPD and RFLP.

The purification procedure requires no phenol or chloroformextraction or alcohol precipitation, and involves minimal handling. The wholeprocedure takes only 20 minutes after sample preparation.

Note: The kit cannot distinguishdifferent forms of DNA and will not be able to separate mitochondrial DNA fromgenomic DNA.

Features

• High quality ofDNA, OD₂₆₀/OD₂₈₀ of purified DNA is generally 1.8~1.9.
• Fast and effective.Fast and easy processing using a rapid spin-column format.
• Compatible withmany downstream applications such as PCR, restriction digestion and hybridization.
• No phenol/chloroformextraction or ethanol precipitation is required.

MaterialsSupplied by User:

• Microcentrifugecapable of at least 8,000 × g
• Pipets andpipet tips
• Vortexer
• Ethanol(96-100%)
• RNase A (20mg/ml, Optional for RNA-free DNA)
• Water bath forheating at 56°C

Before Starting:

This protocol is designed for purification of total DNA fromfresh or frozen anticoagulated blood. All centrifugation steps are carried outat room temperature (15-25°C)in a microcentrifuge. It is strongly advised that you read this booklet thoroughlybefore starting. BP-10 Column Blood Genomic DNA Purification Kit is designed tobe simple, fast and reliable provided that all steps are followed diligently.Prepare all components, and have the necessary materials as outlined beforestarting.

Proteinase K is supplied in a ready-to-use solution form,but RNase A is not provided in this kit, if RNA-free DNA are required, pleaseprepare RNA solution and see protocol to add the RNA removal step.

Check the Universal Buffer CL for salt precipitation beforeeach use. If necessary, re-dissolve the precipitate by warming the solution at 56°C, then cool back down to roomtemperature before use.

CE Buffer is 10 mMTris-HCl, 0.5 mM EDTA, pH 9.0. Watercan be used as eluate in the final step if EDTA should be avoided for thefollowing applications, but it is not recommended if the pH of water is lessthan 7.0.

Preheat the water bath or rocking platform to 56°C.

Procedures

1. Sample Preparation.
   1. No nucleated: Pipet 20 µl proteinase K into each well on a 96-Well Collection Plate. Add 50-100 µl anticoagulated blood. Adjust the volume to 220 µl with PBS. Continue with step 2.
   2. Nucleated: Pipet 20 µl proteinase K into each well of 96-Well Collection Plate. Add 5–10 µl anticoagulated blood. Adjust the volume to 220 µl with PBS. Continue with step 2.
2. Add 200 µl Universal Buffer CL to the sample and covered with sealing film, mixthoroughly by vortexing. Incubate at 56°Cfor 10 min.
   1. Note: If RNA-freegenomic DNA is required, add 20 µl RNase A (20 mg/ml), mix by vortexing, andincubate for 2 min at room temperature before continuing with step 3.
3. Add 200 µl ethanol (96-100%) and covered with a new Sealing film, mixthoroughly by vortexing.
   1. Note: Spin the 96-Well Collection Plate atroom temperature before discard the Sealing film.
4. Transfer the mixture from step 3 (including any precipitate) into the BP-1096-Well Plate placed in a new 96-Well Collection Plate. Centrifuge at 5,000 x g (6,000 rpm) for 1 min. Discard theflow-through.
5. Add 500 µl CW1 Solution, and centrifuge for 1 min at 5,000 x g (6,000 rpm). Discard the flow-through.
   1. Note: Check thelabel to ensure CW1 Solution was diluted with ethanol.
6. Add 500 µl CW2 Solution, and centrifuge for 1 min at 5,000 x g (6,000 rpm). Discard the flow-through.
   1. Note: Check thelabel to ensure CW2 Solution was diluted with ethanol.
7. Place the empty BP-10 96-Well Plate in the 96-Well Collection Plate andcentrifuge for an additional 2 min at 5,000 xg (6,000 rpm) to dry the BP-10 membrane. Discard flow-through and transferthe BP-10 96-Well Plate to a 96-Well Storage Plate.
   1. Note: It isimportant to dry the membrane of the BP-1096-Well Plate, sinceresidual ethanol may interfere with subsequent reactions. This centrifugationstep ensures that no residual ethanol will be carried over during the followingelution.
8. Add 50-100 µl Buffer CE directly onto the center part of BP-10 96-Well Platemembrane. Incubate at room temperature for 1 min, and then centrifuge for 1 minat 5,000 x g (6,000 rpm) to elute the DNA.
   1. Note1: Warm the Buffer CE to 60°Cwill increase the elution efficiency
   2. Note 2: Elution with more than 100 µl (e.g. 200 µl) increases the DNA yield, but the concentration will be lower.
   3. Note 3: For maximum DNA yield, repeat elution once as described in this step.
   4. Note 4: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate.
   5. Note 5: For maximum DNA concentration, use the eluate in the microcentrifuge tube for the second elution step.