FastDNA^® Protocol
Revision No. 6540-999-6H01W

The FastDNA Kit is shipped and stored at ambient temperature.

The FastDNA Kit is for use with the FastPrep Instrument and purifies PCR-ready Genomic DNA from plant and animal tissue, cultured cells, bacteria, yeast, whole insects, etc., in less than 30 minutes. The kit consists of three general classes of components:

A. Lysing Matrix

Lysing Matrix tubes include a 1/4" Ceramic Sphere and Garnet Matrix pre-loaded in 2.0 ml tubes. 1/4" Ceramic Cylinders are included in a separate package. Depending on the difficulty of lysing the samples, the following matrix combinations can be made. See the Matrix Comparison Table on the next page as a guideline for choosing one of the four combinations for a particular sample type. Yield and size of the resulting DNA can be optimized by varying the Lysing Matrix Combination, the speed of the FastPrep, and the amount of time that samples are processed.

B. Sample-Specific Cell Lysis Solution (CLS)

• For plant tissues: CLS - VF + PPS (Protein Precipitation Solution)
• For bacteria and animal cells/tissues, from soft to hard: e.g. cultured cells, insects, bone: CLS - TC
• For yeast, algae, and fungi: CLS - Y

C. Purification and Elution Reagents

• Binding Matrix Solution
• SEWS-M (Salt Ethanol Wash)
• DES (DNA Elution Solution is ultra-pure water)
• BBS (Gel Loading Dye)

1. Sample Processing: Choose appropriate:
   1. Lysing Matrix.
   2. CLS.
   3. Sample size.
2. Homogenize in FastPrep Instrument.
3. Centrifuge to pellet debris; transfer supernatant to new tube.
4. Add Binding Matrix; wash.
5. Elute.

Lysing MATRIX Comparison Table

Sample	Lysing Matrix Comb.	Sample Weight	Yield	DNA Size	OD 260/280
Mature	1	100 mg	VF	2.0 µg	20 kb	1.6
Orange	2	100 mg	VF	2.5 µg	20 kb	1.9
Leaf	3	100 mg	VF	3.0 µg	20 kb	1.9
(Citrus aurantium)	4	100 mg	VF	4.5 µg	20 kb	1.8
Mouse	1	60 mg	TC	20 µg	22 kb	1.8
Liver	2	60 mg	TC	24 µg	18 kb	1.7
(C57 Black6 SJ)	3	60 mg	TC	33 µg	18 kb	1.7
	4	60 mg	TC	34 µg	15 kb	1.6
Mouse	1	20 mg	TC	2.5 µg	17 kb	1.9
Tail	2	20 mg	TC	1.8 µg	16 kb	1.9
(C57 Black6 SJ)	3	20 mg	TC	2.0 µg	13 kb	2
	4	20 mg	TC	3.3 µg	11 kb	1.8
Yeast	1	28 mg	Y	10 µg	22 kb	1.7
(Saccharomyces Cerevisiae)	2	28 mg	Y	10 µg	22 kb	1.9
	3	28 mg	Y	10 µg	22 kb	2
	4	28 mg	Y	10 µg	18 kb	2
Bacteria	1	16 mg	TC	27 µg	23 kb	2
(E. coli)	2	16 mg	TC	23 µg	22 kb	1.9
	3	16 mg	TC	25 µg	19 kb	2
	4	16 mg	TC	28 µg	16 kb	2
Note: In each case the FastPrep speed setting was 5 for 10 seconds.

Mature Orange Leaf	Mouse Liver	Mouse Tail
Lane 1: Lambda-Hind III marker Lane 2: Homogenization with Matrix (1) Lane 3: Homgenization with Matrix (2) Lane 4: Homogenization with Matrix (3) Lane 5: Homogenization with Matrix (4)	Saccharomyces cerevisiae	E. coli with plasmid

1. 1. Sample Processing:
   1. Prepare appropriate Lysing Matrix for the sample to be processed. Depending on the level of difficulty in homogenizing a particular sample, the Lysing Matrix in the tubes may be altered (see Introduction, A. Lysing Matrix and Lysing Matrix Comparison Table).
   2. Choose appropriate CLS and add to tube with Lysing Matrix: Plant Samples: Add 800 µl CLS-VF and 200 µl PPS to a tube containing sample and Lysing Matrix. For bacteria and animal cells/tissues, from soft to hard: e.g. cultured cells, insects, bone: Add 1 ml CLS-TC to a tube containing samples and Lysing Matrix. Algae, Fungi, and Yeast: Add 1 ml CLS-Y to a tube containing samples and Lysing Matrix.
   3. Choose appropriate sample size: Samples consist of up to 200 mg of tissue or a 200 µl suspension of cells in water or isotonic saline solutions. [For single cells grown in suspension (bacteria, yeast, algae, tissue culture cell, e.g.): Centrifuge a sufficient volume of culture to provide a pellet size of 50-100 mg wet weight or up to 109 bacteria, 108 yeast/algae, 107 mammalian cells. Resuspend pellets in 100 µl of water or isotonic saline to give a maximum suspension volume of 200 µl and add to tube]. Some tissues have high levels of nuclease activity and require additional measures to prepare DNA. Plants that contain high levels of phenolics may require the addition of 0.1-1% polyvinylpyrolidone (PVP) or 25 mg of polyvinylpolypyrolidine (PVPP, Sigma Chemical). The addition of DTT and sodium thiosulfate to 10 mM decreases the formation of brown discoloration; 1-3% beta-mercaptoethanol may be required in extreme cases.




Important: The volumes are calculated to leave a minimum air space of approximately 0.25 cc. If less air space is present, there is a likelihood of sample loss due to tube failure or deformation around the cap allowing sample to bubble out; this is caused by an increase in pressure with temperature increase during FastPrepª runs. The presence of 0.25 cc of air space in the tube is sufficient to prevent sample loss during routine FastPrep runs.




2. Homogenize in FastPrep Instrument.
   

   For all applications, place tube in FastPrep Instrument and process for 5-30 seconds at speeds from 4.0-5.0 for all applications. Speeds above 5.0 may damage FastPrep tubes and result in loss of your sample.





Homogenization of hard or fibrous tissues can be facilitated by first placing the tissue between two weigh boats or pieces of parafilm and smashing with a hammer just prior to FastPrep processing.





Additional processing time may be required for cartilage, some leaves, and other fibrous or dried samples. Ice-chill tubes for 1-2 minutes after each 30 second processing cycle.





Note: To improve chances of optimized recovery of DNA from extremely dry samples, like bone, an incubation period of 15 minutes to 2 hours is recommended after processing in the FastPrep Instrument.




3. Centrifuge to pellet debris: Spin in microcentrifuge for 5 minutes at 14,000 x g to pellet protein and cell debris. Transfer 600 µl of the supernatant to a clean microcentrifuge tube.




Note: Extending spin to 15 minutes can enhance elimination of excessive debris from large samples or from cells with complex cell walls. Warning: Please check that tubes are balanced by weight and that the bottom or side of the tubes will not scrape the wall of your microcentrifuge to avoid rapid loss of sample.



For improved convenience, the optional SPIN Protocol on the next page uses steps 4a and 5a (instead of 4 and 5 below).


4. Add 600 µl of Binding Matrix; mix gently, and incubate for 5 minutes at room temperature. Spin for 1 minute; discard supernatant. Gently resuspend pellet with 500 µl SEWS-M. Spin for 1 minute and discard supernatant. Spin for 10 seconds and remove residual liquid with a small bore pipet tip.



5. Elute DNA from Binding Matrix by gently resuspending in 100 µl DES followed by a 2-3 minute incubation. Spin for 1 minute at 14,000 x g and transfer supernatant to a new tube. Be careful to avoid transferring particles of Binding Matrix pellet with your DNA sample. [Use of a large bore pipet will reduce the chance of mechanical shearing of the resulting DNA]. DNA is now ready for digestion, electrophoresis, PCR, and any other desired application.

Follow steps 1-3 of the Detailed Protocol and continue with steps 4a and 5a below.

Optional SPIN Modules (See pg 16) are necessary for steps 4a and 5a.

4a. Add 600 µl Binding Matrix; mix gently, and incubate for 5 minutes at room temperature. Pulse spin for 5 seconds to pellet Binding Matrix and discard supernatant. Gently resuspend pellet with 500 µl SEWS-M and transfer suspension to a SPIN Filter. Centrifuge for 1 minute and discard contents of Catch Tube. Centrifuge for 1 minute to "dry" Binding Matrix/DNA complex. Transfer SPIN Filter to a new Catch Tube.

5a. Elute DNA by resuspending Binding Matrix/DNA complex in 100 µl DES. Wait for 2-3 minutes and centrifuge for 1 minute at 14,000 x g to transfer DNA-containing DES to the Catch Tube; discard SPIN Filter. DNA is ready for use without further manipulation.

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