Protocol for BuccalAmpâ„¢ DNA Extraction Kit, QuickExtractâ„¢ DNA by eld18221


									                                                   BuccalAmp™ DNA Extraction Kit
                                         QuickExtract™ DNA Extraction Solution 1.0
                                              Catch-All™ Sample Collection Swabs
                                                 Cat. Nos. BQ0901S, BQ0908S, BQ0916S, QE09050, QEC0925 and QEC091H

The BuccalAmp™ DNA Extraction Kit is a
fast, simple and inexpensive method for col-                           BuccalAmp™ DNA Extraction Kit
lecting and preparing human genomic DNA for                                        Contents
amplification. In contrast, extraction of DNA                        QuickExtract™ DNA Extraction Solution
from blood samples involves considerable ex-                         1.0 & Catch-All™ Sample Collection
pense, patient distress and special handling of                      Swabs 15 tubes and 15 swabs per kit
liquid samples to minimize exposure to blood-                        respectively. Sufficient for 15 extrac-
borne pathogens. Buccal cell samples can be                          tions.
collected by individuals without extensive train-
ing, potentially facilitating the participation of                   Cat. No. BQ0901S......................1 Kit
family members in genetic studies.1 Individual,                      Cat. No. BQ0908S.................... 8 Kits
sterile Catch-All™ Sample Collection Swabs                           Cat. No. BQ0916S.................. 16 Kits
are provided in positive-seal, hard plastic
tubes for secure storage and transport from
the site of collection to the site of analysis.                      Also available:
Dry brush samples are stable for up to 1 week                        QuickExtract™ DNA Extraction Solution
at temperatures up to 37oC. Subsequent DNA                           1.0. Sufficient for 100 extractions.
extraction requires only heat treatment to lyse
the epithelial cells and to degrade compounds                        Cat. No. QE09050 .................... 50 ml
inhibitory to amplification.2 Following heat
treatment, sample DNA is ready for PCR.                              Catch-All™ Sample Collection Swabs.
Yields range from 0.5-7 µg of DNA from each                          Sufficient for 25 extractions.
buccal sample. Furthermore, extracted DNA                            Cat. No. QEC0925 .............. 25 swabs
contains MasterAmp™ PCR* Enhancer (with
betaine),+ which substantially improves product                      Sufficient for 100 extractions.
yield and decreases nonspecific product for-
mation with many difficult templates.                                Cat. No. QEC091H............ 100 swabs

S t o r a g e : Upon arrival, store the tubes of
QuickExtract™ DNA Extraction Solution 1.0 at
4oC. Do not subject the Extraction Solution to                  Related Products: The following products are
repeated freeze/thaw cycles. Store the sterile                  also available:
Catch-All™ Sample Collection Swabs at room                      − MasterAmp™ Buccal Swab DNA Extraction
temperature.                                                       Kit
                                                                − MasterAmp™ Buccal Swab DNA Extraction
Quality Control: The BuccalAmp DNA Extrac-                         Solution
tion Kit is function-tested by assaying for the                 − MasterAmp™ Buccal Swab Brushes
production of a PCR product from a human X                      − FailSafe™ PCR PreMix Selection Kit
chromosomal marker (Cooperative Human                           − FailSafe™ PCR System
Linkage Center, marker DXS7132), using a                        − MasterAmp™ Taq, Tth, Tfl and
buccal cell DNA preparation as template.                           AmpliTherm™ DNA Polymerases
                                                                − MasterPure™ Complete DNA and RNA
                                                                   Purification Kit
*,+ , See page 2 for patent information.                        − dNTP Solutions

                                                                                                             Lit. #150

          •   (800) 284-8474   •   (608) 258-3080   •   Fax (608) 258-3088
EPICENTRE                                       BuccalAmp™ DNA Extraction Kit
                                      QuickExtract™ DNA Extraction Solution 1.0
                                           Catch-All™ Sample Collection Swabs

1. Richards, B. et al., (1993) Hum. Molec. Genet. 2, 159.
2. Ruano, G. et al., (1992) BioTechniques 13, 266.
3. Ausubel, F. et al., (eds.) (1995) Current Protocols in Molecular Biology (CD ROM ver. 3.7.5) John
    Wiley and Sons, New York, Appendix 3D.

    Use of Betaine for DNA Polymerase Reactions, including, but not limited to use for PCR or DNA
    Sequencing, is covered by U.S. Patent No. 6,270,962, European Patent No. 0742838, German
    Patent No. DE4411588C1 and other issued or pending applications in the U.S. and other coun-
    tries that are either assigned or exclusively licensed to EPICENTRE. These products are accom-
    panied by a limited non-exclusive license for the purchaser to use the purchased products solely
    for life science research. Contact EPICENTRE for information on licenses for uses in diagnostics
    or other fields.

*   Notice to Purchaser: Disclaimer of License, This product is optimized for use in the Polymerase
    Chain Reaction (PCR) covered by patents outside the U.S. owned by F. Hoffmann-La Roche Ltd.
    No license under these patents to use the PCR process is conveyed expressly or by implication to
    the purchaser by the purchase of this product. Where the PCR process is covered by patents, a
    license to use PCR for certain research and development activities accompanies the purchase of
    certain reagents from licensed suppliers such as EPICENTRE when used in conjunction with an
    authorized thermal cycler, or is available from Applied Biosystems. Further information on purchas-
    ing licenses to practice the PCR process where the process is covered by patents may be ob-
    tained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive,
    Foster City, California 94404 or the Licensing Department, Roche Molecular Systems, Inc., 1145
    Atlantic Avenue, Alameda, California 94501.

                BuccalAmp, QuickExtract, Catch-All, AmpliTherm, MasterAmp, Fail-Safe
                 and MasterPure are trademarks of EPICENTRE, Madison, Wisconsin.

                                                page 2
EPICENTRE                                      BuccalAmp™ DNA Extraction Kit
                                     QuickExtract™ DNA Extraction Solution 1.0
                                          Catch-All™ Sample Collection Swabs

                                 Rapid DNA Extraction Protocol

 1. Label the appropriate number of tubes containing QuickExtract DNA Extraction Solution 1.0.

 2. Thoroughly rinse out the subject’s mouth twice with water. We recommend that subjects abstain
    from drinking coffee before tissue collection. Alternatively, instruct subjects to gently brush the
    inside surface of both cheeks with a toothbrush (without toothpaste); follow with a thorough rins-
    ing of the mouth with water.

 3. Collect tissue by rolling the Catch-All sample collection swab firmly on the inside of the cheek,
    approximately 20 times on each side, making certain to move the brush over the entire cheek. If
    storing or transporting the sample, air dry the swab for 10-15 minutes at room temperature.
    Store the dry swab in the original packaging at 22-37oC for up to one week before extracting the
    DNA. For longer storage, place the dry swabs in the original packaging at -20oC for up to 6
    months. Yield is directly correlated with the starting amount of buccal cells. If yield is not a con-
    cern, use only one swab; if yield must be maximized, use a separate swab for each cheek sur-
    face, and if necessary, use a third swab, collecting tissue from both cheeks.

 4. Place the swab end of the Catch-All sample collection swab into a tube containing QuickExtract
    DNA extraction solution and rotate the brush a minimum of 5 times. Press the brush against the
    side of the tube and rotate the brush while removing it from the tube to ensure most of the liquid
    remains in the tube.

 5. Screw the cap on the tube tightly and vortex mix for 10 seconds. Incubate the tube at 65oC for
    1 minute.

 6. Vortex mix for 15 seconds.

 7. Transfer the tube to 98oC and incubate for 2 minutes.

 8. Vortex mix for 15 seconds.

 9. Quantitate DNA yield by fluorimetry using Hoechst dye 33258 to avoid an over estimation given
    by A260 readings.3

10. Store the DNA at -20oC, or at -70oC for long term storage.

The yield of DNA is usually between 2-14 ng/ml. The QuickExtract DNA Extraction Solution 1.0 con-
tains the MasterAmp PCR Enhancer (with betaine). The presence of this reagent may change the
annealing temperature of a given primer pair. We recommend using 5 ml of extracted DNA in a 50 ml
PCR amplification reaction. For target sequences containing high G+C content or secondary struc-
ture, we recommend using 5-15 ml of the extracted sample.

                                                page 3
EPICENTRE                                                                                                     BuccalAmp™ DNA Extraction Kit
                                                                                                    QuickExtract™ DNA Extraction Solution 1.0
                                                                                                         Catch-All™ Sample Collection Swabs

                                                                         QuickExtract DNA Extraction Protocol
The QuickExtract DNA Extraction Solution, currently available separately or as a component of the
BuccalAmp DNA Extraction Kit, provides an extremely efficient method for extracting PCR-ready
genomic DNA from diverse samples. The following protocol has been used to extract PCR-ready
genomic DNA from HeLa cells, hair follicles, tail snips, bacterial cells and feathers.

1. Label the appropriate number of tubes containing 0.5 ml of QuickExtract Solution.

2. Place one sample in each tube, for example:
    • 104 counted human cervical carcinoma tissue culture (HeLa) cells.
    • a 0.5-1 cm region of a single plucked human hair with follicle.
    • a 0.5-1 cm section of a mouse tail snip, finely diced using a fresh blade.
    • one single E. coli colony picked from a plate.
    • a 0.5-1 cm quill-end of a breast feather that was plucked and stored at 4oC.

3. Vortex mix for 15 seconds.

4. Transfer the tube to 65oC and incubate for 6 minutes.

5. Vortex mix for 15 seconds.

6. Transfer the tube to 98oC and incubate for 2 minutes.

7. Store the DNA at -20oC, or at -70oC for long term storage.

8. Use 5 µl or less of the extracted DNA for each PCR amplification (see Figure 1).

Figure 1. Genomic DNA extracted from a variety of tissues or cells using the QuickExtract DNA
          Extraction Solution were consistently amplified using the FailSafe PCR System.

      M              1                  2            3                        4                  5             6
                                                                                                                                  Lanes 1-3, human β-globin;
                                                                                                                                  Lane 4, mouse GAPDH;
                                                                                                                                  Lane 5, E. coli 16 S ribosomal RNA gene;
                                                                                                                                  Lane 6, avian viral sequence
                                      HeLa Cells

                                                   Human Hair Follicle

                                                                                              E. coli Colony
    DNA Ladder

                 Human Buccal Cells

                                                                          Mouse-tail Tissue

                                                                                                               Avian Feather

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