Download G NOME® Whole Blood DNA Isolation Kit

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BIO 101 Protocol
G NOME® Whole Blood
DNA Isolation Kit
2011-200
2011-400
2011-600
10 preps
25 preps
100 preps
• Efficiently Isolate High Molecular Weight Genomic
DNA from Whole Blood
• DNA is Suitable for Restriction Enzyme Digestion
or PCR Amplification
• The Procedure Does Not Use Organic Extractions
Shipping & Storage:
The kit is shipped at ambient temperature. However, upon
arrival store RNase MIXX and Protease MIXX at 4°C. A few
days at ambient temperature does not affect activities of these
reagents. The Protease MIXX is a suspension. Cloudiness or
visible aggregates may appear and can increase with time but
do not affect activity.
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We have you time in mind!”
1070 Joshua Way, Vista CA 92083, USA
Revision #: 2011-999-7H02P
Tel: (760) 598-7299 • Fax (760)598-0116
E-mail: [email protected]
Website: www.bio101.com
For technical support call (800) 424-6101
2
G NOME® Whole Blood DNA Isolation Protocol
Kit Components ......................................................... 3
2011-200 (10 preps) ...............................................................3
2011-400 (25 preps) ...............................................................3
2011-600 (100 preps) .............................................................3
Introduction................................................................4
G NOME® Whole Blood DNA Isolation Protocol ....... 5
Product Use Limitation & Warranty .......................... 8
General Information ...............................................................8
Call (800) 424-6101 for Technical Support
3
Kit Components
2011-200 (10 preps)
Name
Volume
Cell Lysis/Denaturing Solution
1ml
RNase MIXX
0.6ml
Protease MIXX
0.3ml
“Salt-out” Mixture
6ml
Cell Suspension Solution
20ml
Ace Blood Solution
100ml
Catalog #
2010-201
2010-202
2010-203
2010-204
2010-205
2011-206
2011-400 (25 preps)
Name
Volume
Cell Lysis/Denaturing Solution 2.5ml
RNase MIXX
1.40ml
Protease MIXX
0.8ml
“Salt-out” Mixture
15ml
Cell Suspension Solution
50ml
Ace Blood Solution
250ml
Catalog #
2010-401
2010-402
2010-403
2010-404
2010-405
2011-406
2011-600 (100 preps)
Name
Volume
Cell Lysis/Denaturing Solution
10ml
RNase MIXX
5.5ml
Protease MIXX
3ml
“Salt-out” Mixture
60ml
Cell Suspension Solution
200ml
Ace Blood Solution
1000ml
Catalog #
2010-601
2010-602
2010-603
2010-604
2010-605
2011-606
Call (800) 424-6101 for Ordering Information
4
Introduction
The G NOME® Whole Blood DNA Isolation Kit is used to efficiently
isolate high molecular weight genomic DNA from whole blood. Each preparation yields up to 100µg of genomic DNA from 4 ml of whole blood; preparation
sizes can be scaled up or down. The DNA is suitable for restriction enzyme
digestion or PCR amplification usually 2-3 hours after cell lysis; much of the
time involved is spent during a protease incubation. The procedure does not use
organic extractions.
The G NOME® Whole Blood DNA Isolation Kit is available in 3 sizes to
suit your needs: 10, 25, and 100 preparations/kit. The kit contains Ace Blood
Solution to eliminate hemoglobin/heme/porphyrins (that can inhibit PCR
reactons) by lysing cell membranes. Nuclei are lysed with Cell Lysis/Denaturing Solution. The kit utilizes RNase MIXX to eliminate RNA immediately after
lysis, and Protease MIXX to degrade cellular proteins. This is followed by a
proprietary “salting-out” technique which precludes the need for phenol,
chloroform, or other organic extractions. The DNA is precipitated with ethanol,
pelleted by centrifugation and dissolved in TE.
Call (800) 424-6101 for Technical Support
5
G NOME® Whole Blood DNA Isolation
Protocol
Note: The following protocol is based on starting with 2 ml of whole
blood or 500 µl of packed cells. The protocol can be altered
accordingly for larger or smaller volumes.
Expected yield: 20-25 µg DNA/ml whole blood depending on
nucleated cell count.
1.
Centrifuge the whole blood for 5 minutes at 500xg to sediment the
cells. Carefully aspirate most of the plasma being careful not to disturb
cell pellet.
2.
Add 2.5x volumes of Ace Blood Solution and mix gently by inverting
the tube. Centrifuge 15 minutes at 1000-1500xg. Decant the supernate.
Repeat wash with similar volume (2.5x) of the Ace Blood Solution.
Result should be slightly pink to white nuclear pellet.
3.
Bring cells to a final volume of 1.85 ml in Cell Suspension Solution.
(Use a 15 ml clear plastic tube for efficient mixing). Mix until the
suspension appears homogeneous.
4.
Add 50 µl of RNase MIXX, mix thoroughly.
5.
Add 100 µl of Cell Lysis/Denaturing Solution**, mix well.
**If a precipitate forms in your stock solution, heat to 60° C to
dissolve before using.
6.
Incubate at 55°C for 15 minutes.
7.
Add 25µl Protease MIXX, mix thoroughly. (Note: If precipitate is
visible in Protease MIXX suspension, pulse spin and use 25µl of
supernatant.)
8.
Incubate at 55°C for 30 to 120 minutes. (The longer time will result in
minimal protein carry over and will also allow for substantial reduction
in protease activity.)
Call (800) 424-6101 for Ordering Information
6
9.
Add 500µl “Salt Out” Mixture, mix gently yet thoroughly. Divide
sample into two 1.5ml tubes. Refrigerate at 4°C for 10-20 minutes to
produce a visible precipitate.
10. Centrifuge for 10 minutes at high speed (12,000xg) in a
microcentrifuge. Carefully collect the supernate, avoid the pellet. If a
precipitate remains in the supernate, centrifuge again until it is clear.
Transfer the supernate to a 15ml (or larger) translucent poly-propylene
tube, (eg., Falcon 2059).
11. To this supernate, add 2 ml TE buffer and 8 ml of 100% ethanol.
Gently mix the solution by inverting the tube and hold it at room
temperature for 5-10 minutes. Centrifuge for 15 minutes at 10001500xg. Carefully decant or aspirate off the ethanol phase and eliminate the excess ethanol by air drying the DNA.
12. Dissolve the genomic DNA in sterile (autoclaved) TE. (10 mM Tris pH
7.5, 1 mM EDTA)
Call (800) 424-6101 for Technical Support
7
Notes
Call (800) 424-6101 for Ordering Information
Product Use Limitation & Warranty
Unless otherwise indicated, this product is for research use only. Purchase
of BIO 101 products does not grant rights to reproduce, modify, repackage the
products or any derivative thereof to third parties. BIO 101 makes no warranty
of any kind, expressed or implied, including merchantability or fitness for any
particular purpose, except that the products sold will meet our specifications at
the time of delivery. Buyer's exclusive remedy and BIO 101's sole liability
hereunder shall be limited to, at our option, product credits, refund of the
purchase price of, or the replacement of all material(s) that does not meet our
specification. By acceptance of the product, Buyer indemnifies and holds BIO
101 harmless against, and assumes all liability for the consequence of its use or
misuse by the Buyer, its employees or others, including, but not limited to, the
cost of handling. Said refunds or replacement is conditioned on Buyer notifying
BIO 101 within thirty (30) days of receipt of product. Failure of Buyer to give
said notice within thirty (days) shall constitute a waiver by the Buyer of all
claims hereunder with respect to said material(s).
General Information
BIO 101 is a pioneer in developing kits for molecular biology research. We
introduced the GENECLEAN® Kits in 1986 and have since been manufacturing
products to bring convenience into your research. Our goal is to make your life
easier by simplifying the complexities of lab work.
Technical Support and Ordering Information
(800) 424-6101 (Toll Free - Continental USA)
(760) 598-7299 (Outside USA)
(760) 598-0116 (Fax line)
[email protected]
[email protected]
Office Hours:
6:30 am - 6:00 pm P.S.T. (Mon - Fri)
Mailing Address:
BIO 101, Inc.
P.O. Box 2284
La Jolla, CA 92038-2284 USA