About Xpedition™ Tissue & Insect DNA MiniPrep
Note: This product will be discontinued within the next 3 months (November 30th, 2016) or while supplies last. Please refer to ZR Tissue & Insect DNA MiniPrep (D6016) for the replacement product; the Xpedition Stabilization/Lysis Solution (D6202-1-40) can be purchased separately.Degradation and contamination of biological samples have been obstacles to scientific study, and may be particularly problematic in highly sensitive molecular-analysis techniques (e.g., PCR of low copy DNA). Use of cryogenic freezing methods for environmental/forensic sample preservation may often be too impractical to be employed. The Xpedition™ Tissue & Insect DNA MiniPrep, when used with a portable bead beating device [i.e., Xpedition™ Sample Processor (XSP), pg. 6], allows the researcher/investigator to "Take the Lab to the Field" and to the site of sample collection. The Xpedition™ Tissue & Insect DNA MiniPrep can be used for the isolation of inhibitor-free DNA from tough-to-lyse tissues and small organisms (insects). Samples from remote locations can be processed at the site of collection using the provided ZR BashingBead™ Lysis Tubes and a XSP (available separately, Cat. No. S6020, pg. 6). Sample processing occurs in a unique lysis/stabilization solution that effectively preserves DNA in tissue lysates for subsequent storage and transport. Later, when convenient with the researcher, the kit's Fast-Spin column technologies are used for the isolation of inhibitor-free DNA from the lysates. Eluted DNA is ideal for PCR for genotypic analysis including metagenomics, barcoding, phylogenetics, forensics, etc. An overview of the procedure is shown below.
|Format||Bead Beating, Spin Column|
|DNA Recovery||Typically, up to 25 µg total DNA is eluted into 100 µl (25 µl minimum) DNA Elution Buffer per sample.|
|Equipment||Microcentrifuge, vortex, cell disrupter/pulverizer (optional)|
|DNA Size Limits||Capable of recovering genomic DNA up to and above 40 kb. In most instances, mitochondrial DNA and viral DNA (if present) will also be recovered.|
|Sample Sources||Samples (n ≥ 1 and ≤ 50 mg) of fresh, frozen, or stored insects including: mosquitoes, bees, lice, ticks, D. melanogaster, etc. Also, compatible with fresh or frozen mammalian tissues as well as cultured cells, and whole blood.|
|DNA Yield||Expected yields can range from 1 - 5 µg DNA per mg specimen sampled. For mammalian tissues, yields are 1 - 3 µg DNA per mg of skeletal, heart and brain tissues and 3 - 5 µg DNA per mg of liver, kidney, and lung tissues. Whole blood will yield from 3 - 7 µg DNA per 100 µl.|
|Purity||High quality DNA is eluted with DNA Elution Buffer that is well suited for PCR amplification, endonuclease digestion, etc. A260/A280 <1.8|
Add specimen(s) to a ZR BashingBead™ Lysis Tube. Add 750 µl Xpedition™ Lysis/Stabilization Solution to the tube.
Note: The Xpedition™ Lysis/Stabilization Solution can be added to the ZR BashingBead™ Lysis Tube ahead of time, prior to sample addition.
Generally, no more than 50 mg tissue should be sampled, for larger samples will exceed the DNA binding capacity of the spin column (See Specifications on page 1). Up to 400 µl of whole blood or up to 8.5 x106 cells suspended in 200 µl PBS can also be sampled.
Secure in a bead beater fitted with a 2 ml tube holder assembly (e.g., Xpedition™ Sample Processor for processing at the site of sample collection) and process for a minimum of 30 seconds. Store/transport samples. Proceed to Step 3 when convenient.
Centrifuge the ZR BashingBead™ Lysis Tube in a microcentrifuge at ≥10,000 x g for 1 minute.
Transfer up to 400 µl supernatant to a Zymo-Spin™ IV Spin Filter (orange top) in a Collection Tube and centrifuge at 7,000 x g (~ 7,000 rpm) for 1 minute.
Add 1,200 µl of Genomic Lysis Buffer to the filtrate in the Collection Tube from Step 4 and mix.
Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IIC Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
Discard the flow through from the Collection Tube and repeat Step 6.
Add 200 µl DNA Pre-Wash Buffer to the Zymo-Spin™ IIC Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute.
Add 500 µl g-DNA Wash Buffer to the Zymo-Spin™ IIC Column and centrifuge at 10,000 x g for 1 minute.
Transfer the Zymo-Spin™ IIC Column to a clean 1.5 ml microcentrifuge tube and add 50 µl (25 µl minimum) DNA Elution Buffer directly to the column matrix. Centrifuge at 10,000 x g for 30 seconds to elute the DNA.