Promega质粒小抽简要操作手册(中文版)

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?Solution Preparation

Before lysing cells and purifying DNA, prepare the Column Wash Solution by adding ethanol. Cap tightly after addition. See Technical Bulletin #TB374 for detailed instructions.

Centrifuge.

Centrifuge.

Transfer PureYield?Minicolumn to a clean 1.5ml microcentrifuge tube.

Add Elution Buffer or water.

7475M A

Lyse cells.Neutralize.

Transfer supernatant to PureYield? Minicolumn.

Centrifuge.

Centrifuge to elute DNA.

Place PureYield? Minicolumn in a Collection Tube. Centrifuge.

Add Endotoxin Removal Wash.

Add Column Wash Solution.

Alternative Protocol for Larger Culture Volumes

1.Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.

2.Add an additional 1.5ml of bacterial culture to the same tube and repeat Step 1.

3.Add 600μl of TE buffer or water to the cell pellet, and resuspend completely.

4.Proceed to Step 2 of the standard protocol above.

DNA Purification by Centrifugation

Prepare Lysate

1.Add 600μl of bacterial culture to a 1.5ml microcentrifuge tube.

N o t e :For higher yields and purity use the alternative protocol below to harvest and process up to 3ml of bacterial culture.2.Add 100μl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.3.Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting.

4.Centrifuge at maximum speed in a microcentrifuge for 3 minutes.

5.Transfer the supernatant (~900μl) to a PureYield? Minicolumn without disturbing the cell debris pellet.

6.Place the minicolumn into a Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.

7.Discard the flowthrough, and place the minicolumn into the same Collection Tube.Wash

8.Add 200μl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds.

9.Add 400μl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.Elute

10.Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30μl

of Elution Buffer or nuclease-free water directly to the minicolumn matrix. Let stand for 1 minute at room temperature.

11.Centrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge

tube, and store eluted plasmid DNA at –20°C.

For complete protocol information see Technical Bulletin #TB374, available at: w

w w w .p r o m e g a .c o m /t b s

Prepare Lysate

1.Transfer 1.5ml of culture to a 1.5ml microcentrifuge tube

N o t e :If you wish to process larger volumes of bacterial culture (up to 3ml) use the alternative protocol provided below.2.Centrifuge at maximum speed in a microcentrifuge for 1 minute.3.Remove and discard medium.

4.Resuspend the cell pellet in 600μl of TE buffer or water.

5.Add 100μl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.

6.Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting.

7.Centrifuge at maximum speed in a microcentrifuge for 3 minutes. Place a PureYield? minicolumn on a Luer-Lok ?adapter of a VacMan ?or VacMan ?Jr Laboratory Vacuum manifold

8.Transfer the supernatant (~900μl) into a PureYield? Minicolumn.9.Apply vacuum pulling the lysate through the column.

Wash

10.Add 200μl of Endotoxin Removal Wash (ERB) to the minicolumn. Allow the vacuum to pull the solution through the column.11.Add 400μl of Column Wash Solution (CWC) to the minicolumn. Allow the vacuum to pull the solution through the column.

Release the vacuum, and remove the PureYield? Minicolumn.Elute

12.Place the column in a 2ml collection tube, and centrifuge at maximum speed in a microcentrifuge for 1 minute.

13.Transfer the minicolumn into a clean 1.5ml microcentrifuge tube, then add 30μl of Elution Buffer or nuclease-free water

directly to the minicolumn matrix. Let stand for 1 minute at room temperature.

14.Centrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.

For complete protocol information see Technical Bulletin #TB374, available at: w

w w w .p r o m e g a .c o m /t b s DNA Purification by Vacuum

Alternative Protocol for Larger Culture Volumes

1.

Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge.2.Discard the supernatant.

3.Add an additional 1.5ml of bacterial culture to the same tube. Repeat Steps 1 and 2.

4.Add 600μl of TE buffer or water to the cell pellet, and resuspend completely.

5.

Proceed to Step 5 of the standard protocol above.

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