AutoGen Protocol - Whole Blood 5ml DNA

{AutoGenFlex 3000}

AGF3000 Frozen Whole Blood Protocol
(AutoGenFlex 3000 + FlexiGene Chemistry from QIAGEN)

Download printable AutoGenFlex 3000 Protocol Sheet (acrobat file)

I. Purpose: To extract genomic DNA from EDTA, or citrate treated frozen human whole blood.

II. Chemical Principal: QIAGEN's FlexiGene Procedure (protease digestion/DNA precipitation method)

III. Protocol Parameters:

A. Sample Volume: 1.0 - 5.0 ml of frozen whole blood or frozen buffy coat prepared from
1.0-5.0ml of whole blood.

B. Maximum Number of Samples: Up to 40 samples per run.

C. Throughput: Up to 80 x 5.0 ml or 40 x 10.0 ml samples per day.

D. Yield: 50 - 210 μg of purified DNA per 5.0ml of frozen whole blood.

E.     Quality: Typical OD_260/280values are 1.70 - 1.90.
                      The DNA is free of contaminants and inhibitors (RNA, protein) and can be used
                      directly in downstream processes such as DNA sequencing, PCR-based assays,
                      restriction digestions and Southern blotting.

IV. Running the Protocol:

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

V. Example of genomic DNA obtained from frozen whole blood:

Example of Genomic DNA obtained from frozen whole blood:

Genomic DNA was isolated on the AutoGenFlex 3000 using the
FlexiGene 5ml frozen whole blood protocol. The starting material was 5.0 ml
of EDTA or citrate treated frozen and thawed whole blood from four different donors with four replicates each. The yield from 5.0 ml whole blood is 50-210 μg
genomic DNA with a high molecular weight of 30–150 kb.

VI. Extraction and Purification Process:

Process Site	Purpose	System Process
1. Automated	Collect white cells	Add Buffer FG3*, centrifuge and discard the supernatant.
2. Automated	Lyse white cells	Add Buffer FG1*, centrifuge and discard the supernatant. Repeat.
3. Automated	Digest/lyse nuclei	Add QIAGEN Protease* and Buffer FG2*. Mix, incubate and mix again.
4. Automated	Precipitate DNA	Add R4** or Isopropanol to DNA recovery tube. Transfer supernatant of sample tube into DNA recovery tube, mix, centrifuge and discard the supernatant.
5. Automated	Wash DNA	Add R5** or 70% Ethanol, mix, centrifuge and discard the supernatant.
6. Automated	Resuspend DNA**	Add reagent FG3*, mix, incubate, mix and centrifuge.

* Buffers FG1, FG2 and QIAGEN Protease are available from QIAGEN as FlexiGene DNA AGF3000 Kit (640), Catalog #51297.
** Reagents R4 and R5 are available from AutoGen, Inc. as AutoGenFlex 3000 Blood DNA Finishing Kit, Catalog #AGFXPW.
*** Samples can be vortexed after initial incubation if required.