PROCOTOL 003: DNA Mini-Prep

REAGENTS

Solution I (P1)

Solution II (P2)

Solution III (P3)

100% ethanol

70% ethanol

1X TE, pH 8

 

PROCEDURE                            **SEE NOTES BEFORE STARTING***

 

Pour culture into 1.5 ml microfuge tube—see NOTE 1

Centrifuge for 1 minute at 14K rpm

Dump out or aspirate supernatant taking care not to disturb pellet

Add 100 mL of solution I (also called P1 and stored at 4 C)—see NOTE 2

Using a P200 set to 100 mL, completely resuspend pellet (very important)

Add 200 mL of solution II (also called P2 and stored at RT)

Close tube and mix gently by inverting tube several times, making sure to coat the tube

Let stand at RT for about 5 minutes

Add 150 mL of solution III (also called P3 and stored at 4 C)—see NOTE 3

Close tube and vortex to completely mix contents of tube—see NOTE 4

Set tube on ice for about 10 minutes

Centrifuge for 5 minutes at 14K rpm

Using a P1000 set to 400 mL, remove supernatant without disturbing pellet—see NOTE 5

Transfer supernatant to new tube; discard old tube

Add 1 mL of 100% EtOH at RT and mix by inverting. On ice 10-20 minutes

Centrifuge for 8 minutes at 14K rpm

Working quickly, discard supernatant

Wash pellet by adding 300 mL of 70% EtOH at RT

Centrifuge for 2 minutes at 14K rpm

Working quickly, discard supernatant and set tube upside down on paper towel to dry

When dry, add 50 mL of 1X TE to tube and place tube in 37 C water bath for 30 minutes

 

NOTES

1.     Don't worry about harvesting all the culture. Whatever is left can be used to inoculate a larger culture (e.g. 100 mL) after you've identified the correct clone(s).

 

2.     Solution I (PI) has RNase. Be careful not to spill this solution.

 

3.     When you open tube, contents will be viscous. Take care not to transfer contents of one tube to another.

4.     You should see a white precipitate form—this is the detergent, bacterial DNA and cell debris.

 

5.     There will be a surface skim. Poke through this with pipette tip. It will stick to the outside of the tip and won't contaminate the supernatant.