AutoGen Protocol - Whole Blood DNA

I. Purpose: To extract DNA from EDTA treated whole blood, acid-citrate-dextrose treated whole
blood or heparin treated whole blood.

II. Chemical Principal: Modified GTC (Guanidine thiocyanate) extraction chemistry.

III. Protocol Parameters:

A. Sample Volume: Up to 200μl of whole blood (fresh or frozen).

B. Maximum Number of Samples: Up to 384 (4 of 96 plates) samples per run.

C.    Processing Time: 4.2 hours for 192 samples
                               6.4 hours for 384 samples.
                               Including 60 minutes DNA drying time

D. Yield: 3 - 5 mg of purified gDNA / 200 ml of whole blood.

E.     Quality: Typical OD_260/280values are 1.7-1.8.
                      The DNA can be used directly in downstream processes such as PCR, real time
                      PCR, Southern Blotting and more*.
                      * DNA extracted from heparinized whole blood might not be applicable to some downstream applications.

IV. Running the Protocol:

    A.    Load Reagents and Samples
    B.    Select a Protocol
    C.    Enter Number of Samples
    D. Start the Run

VI. Example of genomic DNA obtained from whole blood:

Genomic DNA was extracted using 3 different methods, and
then the extracted DNA was compared on the gel.

Starting material:200μl of EDTA treated whole blood.
Gel condition: 0.7% agarose gel, 1xTBE, 50V x 1hr

Upper Lanes: Competitor’s manual kit (left hand side)
Manual extraction (right hand side)
Lower lanes: AutoGenprep 965 WB protocol

VII. Extraction and Purification Process:

Process Site	Purpose	System Process
1. Automated	Lyse blood cells	Add Reagent R1* and mix.
2. Automated	Denature protein	Add Reagent R2* and mix.
3. Automated	Precipitate protein	Add Reagent R3* and mix.
4. Automated	Collect DNA	Add Reagent R4*, mix, centrifuge and transfer supernatant to a new plate.
5. Automated	Precipitate DNA	Add Reagent R5*, mix, centrifuge and discard supernatant.
6. Automated	Wash DNA	Add Reagent R6*, mix, centrifuge, and discard supernatant. Repeat 3 times.
7. Automated	Dry DNA	Evaporate residual solvent.
8. Automated	Resuspend DNA	Add Reagent R7*, mix and centrifuge.

* All reagents and consumables are sold as a complete kit for whole blood DNA extraction for the AutoGenprep 965.