GENECLEAN for Ancient DNA
Protocol
Revision No. 1002-999-6A01W

The GENECLEAN For Ancient DNA Kit is designed for isolation of DNA from samples of bone, preserved tissue, or animal byproducts. The reagents are formulated carefully to prevent contamination by contemporary DNA. Each prep size is capable of isolating 20 µg of DNA. The resulting samples are readily amplified by PCR or other methods.

Manual Protocol
Incubation with Proteinase K prior to starting with step 1 has been reported to increase yields of DNA isolated from bone. See Note in appendix.

1. Add 100-500 mg homogenized or powdered sample to 1 ml of DNA Dehybernation Solution* in a nucleic acid-free microcentrifuge tube.




Three different Dehybernation Solutions are supplied with this kit. DeHyb A is a guanidine based solution. A2 (see below**) added to A gives a second version of A. And thirdly, DeHyb B is an aqueous EDTA based solution. With the large diversity of samples and their conditions (degree of preservation and environment), it has proven impossible to predetermine which DeHyb solution will be most efficacious with untried samples. Therefore, a preliminary experiment is recommended: for each sample type try all three in a first experiment: A, A2, and B.






*Dehybernation A2 is an additional detergent that can be used in conjunction with DeHyb A to improve DNA yields. To use DeHyb A2, add 150 µl to 850 µl of DeHyb A just prior to step 2.




2. Incubate at 45-60°C between 2 and 12 hours with mixing (longer incubation times may be necessary).



3. Centrifuge sample at high speed for 5 minutes to pellet particulate material. Transfer supernatant to a new nucleic acid-free tube. Add 300 µl of Ancient DNA GLASSMILK suspension. Incubate at room temperature for 10-30 minutes with mixing.



4. Transfer suspension to a SPIN Filter and Catch Tube. Centrifuge at high speed in microcentrifuge for 1 minute or until liquid is transferred to Catch Tube. (Empty Catch Tube as needed).



5. Add 0.5 ml Salton Wash No. 1 and centrifuge to clean GLASSMILK/DNA complex.



6. Add 0.5 ml Salton Wash No. 2 and centrifuge to clean GLASSMILK/DNA complex. See appendix for optional wash at this point.



7. Add 0.5 ml Ancient DNA Alcohol Wash and centrifuge to empty filter. Repeat. [Important: Be sure to add alcohol prior to first use].



8. Empty Catch Tube and centrifuge for 2 minutes to "dry" pellet in SPIN Filter.



9. Place filter into a DNA-free Elution Catch Tube. Add 50-100 µl of DNA-free Elution Solution. Resuspend pellet by hand or briefly vortex (1-2 seconds; more will damage filter). Centrifuge for 1 minute to transfer eluate to Catch Tube. Optional: Elute a second time to increase yield 10-20%.



10. Remove SPIN Filter and discard. DNA is ready to use in amplification reaction without further manipulation.

1. Add 100-500 mg sample (slightly granular form, whole chunks will not homogenize well) to 1 ml of DNA Dehybernation Solution* in the Homogenization Matrix/Tube. (5 are included in each kit as samples. To order BioPulverizer tubes separately, see pg 68).



2. Vortex for 5 minutes at full speed.



3. Incubate at 45-60°C between 2 and 12 hours with mixing (longer incubation times may be necessary for older or very hard samples).



4. Centrifuge sample at high speed for 5 minutes to pellet particulate material. Transfer supernatant to a new nucleic acid-free tube. Centrifuge again for 3 minutes (to remove any remaining particulates). Transfer supernatant to a new nucleic acid-free tube.



5. Add 300 µl of Ancient DNA GLASSMILK suspension. Incubate at room temperature for 10-30 minutes with mixing. Continue with step 4 in manual protocol.

Protocol for Use with FastPrep FP120 Instrument.
See appendix for note on preincubation with Proteinase K.

1. Add 100-500 mg sample (slightly granular form; whole chunks will not homogenize well) to 1 ml of DNA DeHybernation Solution* in a 2ml BioPulverizer Tube.



2. Process at a setting of 6 in FastPrep FP120 for 5-45 seconds to homogenize sample. Softer samples require shorter times and/or lower settings.
3. Continue with step 3 in the previous protocol above.

* There is an additional detergent supplied with this kit that can be used in conjunction with DeHyb A. In certain applications, this detergent (DeHyb A2) has resulted in improved DNA yields. If you choose to use DeHyb A2, then the amount of DeHyb A should be decreased to 850 ml, and 150 ml of DeHyb A2 must be added just prior to incubation or homogenization (vortexing or FastPrepping).

Washing the DNA/Ancient DNA GLASSMILK pellet with 300 µl of a 1:1 solution of acetone: ethanol prior to step 7 will enhance purity of DNA if subsequent reactions are not working properly.

To facilitate access by the chemical denaturants contained in Dehyb Solutions to cellular materials, a preincubation with Proteinase K is suggested. The following protocol was submitted by a customer.

Overnight Soaking Solution

1. Samples were rotated and incubated at 37° C for 12-15 hours.



2. 1 ml of DeHybernation Solution A was added to each sample and rotated for 2-4 hours at 60°C.



3. Samples were spun in centrifuge to pellet particulate material.



4. Supernatant was transferred to a clean tube and 1.2 ml of Ancient DNA GLASSMILK and 3.0 ml of DeHybernation Solution A were added.



5. Samples were rotated for 2 hours at 35-40°C.



6. Samples were centrifuged at 4,000 rpm to pellet Ancient DNA GLASSMILK. Supernatant was discarded.



7. 0.5 ml of Salton Wash No. 1 was added to resuspend pellet, which was then transferred to a SPIN Filter.



8. The protocol, starting with Step 6 (of Manual Protocol), was followed from this point forward.