AutoGen Protocol - ES Cells DNA- High Throughput

{AutoGenprep 965}

I.     Purpose: To extract genomic DNA from ES cells

II.   Chemical Principal: Proteinase K / non-phenol extraction method.
                                    *Phenol extraction protocol is also available.

III. Pretreatment of ES cells (confluent culture in 24-well plates):

         1. Aspirate and dispose of the culture media.
         2. Wash ES cells with EDTA-PBS buffer.
         3. Add 0.1ml of [0.25%Trypsin / 0.1%EDTA / PBS] solution in the wells.
         4. Incubate the plates at 37°C for several minutes.
         5. Pat the side of plates to get the cells released from the plates.
         6. Transfer the resuspended cells into 96 deep well plates.
         7. Load the 96 deep well plates onto the AutoGenprep 965/960.
         8. Run [Digest] Protocol*. The AutoGenprep 965/960 automatically adds 0.15ml of Reagent M2 (Tissue
             Digestion Solution 2) and 0.05ml of Reagent M1 (Tissue Digestion Solution 1), containing the pre
             dissolved Proteinase K at the concentration of 1.0mg/ml**) into the plates and mixes them.
         9. Remove the plates from the 965/960 and incubate overnight at 60-65°C.

             * Step 8 and 9 can be done manually.

IV. Protocol Parameters:

         A. Sample Volume: Resuspended confluent-cultured ES cells in 24 well plate (0.1ml)

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

         C. Processing Time: 2.5 hours for 192 samples (2 plates)
                                             4.0 hours for 384 samples (4 plates)
                                             This includes a 20 minutes drying period

         D. Yield: Approx. 10 mg of genomic DNA / 1 x 106 cultured cells
                         The actual yield may vary depending on condition and concentration of starting samples.

         E. Quality: OD260/280 values are 1.70 - 1.85
                            OD230/260 values are < 0.4
                            The DNA can be used directly in downstream applications such as fluorescent
                            DNA sequencing, PCR, restriction enzyme digestions and more.

V. Running the Protocol:

         A. Load Reagents and Sample Plates
         B. Select [Extraction] Protocol
         C. Enter Number of Samples
         D. Start the Run

VI. Example of the extracted genomic DNA on the AutoGenprep 965/960:

Gel electrophoresis of the extracted genomic DNA

          Gel: 0.7% agarose, TBE
          Condition: 50V x 60 min.
          Sample: 5μl
          M: λ/Hind III marker

VII. Extraction Process:

Process Site	Purpose	System Process
1. Automated or Manual	Digest Tissues	Dissolve Pro K with Reagent M1, and add this combined solution with Reagent M2 to samples, and incubate overnight at 60-65°C.
2. Automated	Remove RNA	Add Reagent R8 and mix.
3. Automated	Remove Protein and Cellular Debris	Add Reagent R3, mix, centrifuge to pellet debris and transfer supernatant to new plate.
4. Automated	Precipitate DNA	Add Reagent R4, mix, precipitate DNA, centrifuge and discard supernatant.
5. Automated	Wash DNA	Add Reagent R5/6/7, mix, centrifuge, and discard supernatant, repeat.
6. Automated	Dry DNA	Dry DNA by heating.
7. Automated	Resuspend DNA	Add Reagent R9 and mix.