PLASMID ISOLATION AND ANALYSIS Part II

Plasmid Purification and Isolation

PLASMID ISOLATION AND ANALYSIS: Part II

Pellet 2 ml of overnight cell suspension by adding 1 ml (20 drops) of overnight culture suspension into a 1.5 ml reaction tube. Spin for 1 minute at 10,000 rpm in a microcentrifuge (MicroV).

Plasmid Purification and IsolationStep 1

PLASMID ISOLATION AND ANALYSIS: Part II

A pellet should form in the bottom of the tube. Drain off theliquid into a designated container and gently tap the tube on apaper towel to remove the excess broth. Repeat the procedure with a second 1 ml volume of overnight culture suspension.

Plasmid Purification and IsolationStep 1 (cont.)

PLASMID ISOLATION AND ANALYSIS: Part II

Using a small needle pipette, completely resuspend the cell pellet by adding the entire contents of the tube marked “Cell Resuspension Solution”(P1). Mix the contents by finger flicking the tube or pipetting several times. No visible clumps of bacteria should remain.

Plasmid Purification and IsolationStep 2

PLASMID ISOLATION AND ANALYSIS: Part II

Use a small needle pipette to add the contents of the tube marked “Cell Lysis Solution”(P2) to the Resuspension tube and mix by inverting the tube 4 times. The cell suspension should appear clear.

Plasmid Purification and Isolation

Step 3

PLASMID ISOLATION AND ANALYSIS: Part II

Use a small needle pipette to add the contents of the tube marked “Neutralization Solution” (N3) to the Resuspension tube and mix by inverting the tube 4 times.

Plasmid Purification and IsolationStep 4

PLASMID ISOLATION AND ANALYSIS: Part II

Centrifuge the tube containing the lysate at 10,000 rpm for 10 minutes.

Plasmid Purification and IsolationStep 5

PLASMID ISOLATION AND ANALYSIS: Part II

While the lysate is in the centrifuge, label two 1.5 ml tubes.

Label the lid and side of one tube “L”.

Plasmid Purification and IsolationStep 6

PLASMID ISOLATION AND ANALYSIS: Part II

Label the lid and side of the second tube “M.” Remove the lidfrom the tube with a scissors. Place the lid back on the tube and hold until Part III, step 7.

Plasmid Purification and IsolationStep 7

PLASMID ISOLATION AND ANALYSIS: Part II

Use the small needle pipette to carefully remove all of the clear lysate/supernate from the miniprep in step 5. Do not remove any of the precipitate. Wipe the end of the pipette with a chem wipe or kleenex to remove any protein/chromosomal DNA precipitate.

Plasmid Purification and IsolationStep 8

PLASMID ISOLATION AND ANALYSIS: Part II

Pipette the clear lysate/supernate into the tube marked “L.”

At this point you can choose to store the tube in the refrigeratoruntil the next day or, if time permits, continue to Part III.

Plasmid Purification and IsolationStep 9