Training Manual Application of Genetics and Biotechnology in ...

1 .a . Plasmid isolation protocol:
Solution 1 :
50mM glucose
25mM Tris.Cl (pH 8.0)
1 OmM EDTA
20pgr'ml RNAse (final concentration)
Solution 2:
0.2N NaOH
1% SDS
Solution 3: 3M potassium acetate (pH 4.8)
Procedure
1. Fill a microcentrifige tube with saturated bacterial culture grown in LB broth
+ antibiotic. Spin tube in microcentrifuge for 1 minute. Dump supernatant
and drain tube briefly on paper towel.
2. Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as
possible using disposable tip.
3. Add 0.2 ml Solution 2, cap tubes and invert five times gently. Let tubes sit in
ice for 2-3 minutes.
4. Add 0.2 ml ice-cold Solution 3, cap tubes and invert five times gently.
Incubate tubes on ice for 5 minutes.
5. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh micro centrifuge
tube using clean disposable transfer pipette. Try to avoid taking any white
precipitate during the transfer.
6. Fill remainder of centrihge tube with 2 volumes of ethanol. Let tube sit at
room temperature for 2 minutes.
7. Centrifuge tubes for 5 minutes. Pour off supernatant without dumping out the
pellet. Drain tube on paper towel.