AutoGen Protocol - Plasmid DNA

I. Purpose: To extract plasmid DNA from cultures of E. coli.

II. Chemical Principal: Modified alkaline lysis - Non-phenol extraction method.

III. Protocol Parameters:

A. Sample Volume: 0.5 - 1.8 ml liquid culture of E. coli.

B. Maximum Number of Samples: 24 samples per run.

C.    Processing Time: 2.0 hours for 12 samples^*2
                              2.5 hours for 24 samples^*2
                              ^*2 This includes drying and resuspending DNA steps.

D.    Yield*:   3.0 - 5.0 μg of purified DNA per 1.8ml culture of plasmid.
           * Measured by Hoechst 33258
              *Actual yield may vary, depending on the cell density, type of plasmid and host cell.

E.     Quality:   Typical OD_260/280values are ³ 1.75 - 1.85.
                The DNA can be used directly in downstream processes such as
                fluorescence DNA sequencing, PCR and restriction endonuclease digestions.

IV. Running the Protocol:
A.    Load Reagents, Tips and Samples
B.    Select a Protocol
C.    Enter Number of Samples
D.     Start the Run

V. Example of AutoGen purified DNA:

A. AutoGen purified plasmid DNA:

   Gel Electrophoresis Condition:

   Sample: 3 μl of 50 μl resuspended
                 plasmid DNA extracted by
                 the AutoGenprep 240.

   Gel: 0.7% agarose gel,
           1xTEB buffer,
           50V x 1.5hr

B. Sequencing data using AutoGen purified plasmid DNA:

   Sequencing Condition:

   Sample: 50 μl resuspended plasmid (pUC18)
                 from 1ml of E.Coli JM109 culture

   Sequencing kit: BigDye Terminator Cycle Sequencing
                            FS Ready Reaction kit (ABI)

   Primer: Neighbor region of -21M13
   Template: 300-500ng
   Reaction: 96°C, 3min →[96°C, 10sec→50°C,
                   5sec→72°C, 4min] x 25 cycles
   Sequencer: ABI PRISM 310

VI. Extraction and Purification Process:

Process Site	Purpose	System Process
1. Automated	Concentrate Cells	Centrifuge overnight culture. Discard supernatant.
2. Automated	Resuspend Cells, Digest RNA	Add Reagent R1* and mix.
3. Automated	Lyse Cells	Add Reagent R2 and mix.
4. Automated	Remove Protein and Cellular Debris	Add Reagent R3, mix, centrifuge to pellet debris, transfer supernatant to new tube.
5. Automated	Precipitate DNA	Add Reagent R4, mix, precipitate DNA, centrifuge and discard supernatant.
6. Automated	Wash DNA, discard supernatant.	Add Reagent R5, mix, centrifuge.
7. Automated	Evaporate Alcohol.	Centrifuge tube to dry DNA sample.
8. Automated	Resuspend DNA	Add Reagent R6 and mix.
* All reagents are sold as a complete reagent kit for plasmid extraction on the 245T.