Protocol #05-01-001
DNA Purification; Tissue; QIAamp Mini Kit; Centrifuge
Version 1.02
DNA Purification from Tissues using QIAamp DNA Mini Kit
and a Tabletop Centrifuge
Created by: Ion Beldorth on 23 March 2010
Last Edited by: Ion Beldorth on 01 April 2010
The most recent version of this document is located at http://www.xiphophorus.txstate.edu/research/protocols/05/01.html
Appendix A – Complete list of reagents, their storage locations, and important safety precautions.
Appendix B – Recipes for solutions used in this protocol.
Appendix C – Nucleic Acid Extraction: Theory and Practice.
Appendix D – Troubleshooting.
General Information:


Familiarize yourself with pertinent information found in Protocols #01-01-001 (Laboratory
Safety) and #01-01-003 (Guidelines for Molecular Biology).
Centrifugation steps are performed at:
o room temperature (15-25°C)
o maximum speed
Points to consider before beginning:



Use carrier DNA (e.g., poly dA, poly dT, etc.) for low copy number (<10,000) samples.
Transcriptionally active tissues, such as liver and kidney, contain high levels of RNA which will
copurify with genomic DNA. RNA may inhibit some downstream enzymatic reactions, but will
not inhibit PCR. If RNA-free genomic DNA is required, include the RNase A digest as described in
Step 6 of the protocol.
Elute DNA using either Buffer AE (supplied with kit; see Appendix B for in-lab preparation) or
ddH2O (or equivalent).
o Buffer AE is suitable for long-term storage (-20°C), however, ddH2O is preferred where
either the pH (9.0) or presence of EDTA (0.5mM) will affect downstream applications.
o ddH2O is suitable for sensitive downstream applications, however the water must be
greater than pH 7.0 as DNA is subject to degradation by acid hydrolysis (deionized water
becomes acidic due to dissolved atmospheric CO2).
Actions to perform before beginning this Protocol:





Equilibrate Samples and either Buffer AE or ddH2O to room temperature (15-25°C).
Heat a shaking water bath to 56°C for use in Step 3.
Heat a heating block to 70°C for use in Step 5.
Ensure Buffers AW1 and AW2 contain the appropriate amount of ethanol before use.
If Buffer ATL or Buffer AL contains a precipitate, heat to 56°C until dissolved.
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Protocol #05-01-001
DNA Purification; Tissue; QIAamp Mini Kit; Centrifuge
Version 1.02
Begin Protocol
I.
TISSUE COLLECTION
A)
See Protocol #04-01-001 (http://www.xiphophorus.txstate.edu/research/protocols/04/01)
II.
DNA EXTRACTION AND PURIFICATION
Items you will need:
 QIAGEN QIAamp DNA Mini Kit
 Scalpel, #10 blade
 Forceps, fine tip
 1.5 ml Eppendorf Tubes
 Pestles, blue polypropylene
 Ethanol, 96-100%
 RNase A, 10 mg/ml (Optional)
 ddH2O (Optional)
A)
Tissue Lysis
1. Weigh out up to 25 mg of tissue per extraction
If more than 25mg is used, scale up reagents and number of spin columns as appropriate.
2.
3.
a) Chop tissue coarsely (approximately 5 mg/piece) with a scalpel
b) Place tissue in 1.5 ml Eppendorf tube using fine-tip forceps
Add 180 µl of Buffer ATL
a) Gently homogenize, by hand, using a clean blue polypropylene pestle
Add 25 µl Proteinase K
a) Mix thoroughly by vortexing
b) Incubate in a shaking water bath until tissue is completely lysed
 56°C
 1-3 hours
While samples are lysing, prepare a gel for electrophoresis:

Refer to Protocol #05-04-001, Appendix C for selecting buffer and gel
concentration, and Appendix B for preparing buffer and gel.
Continue with remainder of protocol . . .
c)
Spin down briefly
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Protocol #05-01-001
B)
DNA Purification; Tissue; QIAamp Mini Kit; Centrifuge
Version 1.02
DNA Precipitation
Step 4 is optional. Only perform if the presence of RNA will interfere with downstream use.
4.
5.
OPTIONAL: Add 4 µl of RNase A (100 mg/ml)
a) Mix thoroughly, but as briefly as possible, by pulse-vortexing
b) Incubate
 Room temperature
 2 minutes
c) Spin down briefly
Add 200 µl Buffer AL
a) Mix thoroughly, but as briefly as possible, by pulse-vortexing
A white precipitate may form on addition to Buffer AL. The precipitate does NOT interfere
with downstream applications, and may or may not dissolve during incubation at 70°C.
b)
Incubate
 70°C
 10 minutes
While samples are incubating, perform the following steps:
1.
2.
3.
Unpack Spin Columns and place into tube-rack
 If more than 25 mg of tissue was used, scale up number appropriately
a) Label top of Spin Column with Sample ID
Add 2 ml Collection Tubes to the rack
 3x number of extractions
Add 1.5 ml Eppendorf Tubes to the rack
 1x number of extractions
a) Label top of tubes with Experiment ID, sample number, contents (i.e. gDNA),
and your initials.
b) Label side of tubes with Experiment ID, Sample number, contents, elution
liquid, pH (if other than ddH2O), date, concentration and your initials.
Continue with remainder of protocol . . .
6.
c) Spin down briefly
Add 200 µl 100% EtOH
a) Mix thoroughly, but as briefly as possible, by pulse-vortexing
b) Spin down briefly
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Protocol #05-01-001
C)
DNA Purification; Tissue; QIAamp Mini Kit; Centrifuge
Version 1.02
Purification of DNA
7. Apply mixture – including any precipitate – to the QIAamp Mini Spin Column in a 2 ml
collection tube (supplied with kit)
a) Close cap
b) Centrifuge
 1 minute
c) Place column in clean 2 ml collection tube. Discard used tube.
8. Apply 500 µl of Buffer AW1
a) Close cap
b) Centrifuge
 1 minute
c) Place column in clean 2 ml collection tube. Discard used tube.
9. Apply 500 µl of Buffer AW2
a) Close cap
b) Centrifuge
 3 minutes
c) Place column on clean 2 ml collection tube. Discard used tube.
d) Centrifuge
 1 minute
e) Place column on clean, 1.5 ml Eppendorf tube. Discard used tube.
10. Apply 200 µl Buffer AE or ddH2O directly onto the column’s filter
a) Incubate
 Room temperature
 5 minutes
b) Centrifuge
 1 minute
11. Repeat Step 10
 1-2 times
III.
CONCENTRATION AND QUALITY ANALYSIS
A)
See Protocol #05-04-001 (http://www.xiphophorus.txstate.edu/research/protocols/05/04)
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