Miniprep Plasmid DNA Isolation

Plasmid DNA Isolation

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. You will perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol, often referred to as a plasmid "mini-prep," yields fairly clean DNA quickly and easily.

Procedure

1. Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.
2. Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.
3. Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.
   
   • Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH ( = 8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.
4. Add 0.4 ml Solution 2, cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes.
   
   • Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands.
5. Add 0.3 ml ice-cold Solution 3, cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes.
   • Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution.
6. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh microcentrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer. It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.
   
   • This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.
7. Fill remainder of centrifuge tube with isopropanol. Let tube sit at room temperature for 2 minutes.
   
   • Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.
8. Centrifuge tubes for 5 minutes. A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel.
   
   • This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period.
9. Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel.
   
   • Ethanol helps to remove the remaining salts and SDS from the preparation.
10. Allow tube to dry for ~5 minutes. Add 50 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer.

Solutions:

Solution 1:	per 500 ml:
50 mM glucose	9 ml 50% glucose
25 mM Tris-HCl pH 8.0	12.5 ml 1 M Tris-HCl pH 8.0
10 mM EDTA pH 8.0	10 ml 0.5 M EDTA pH 8.0

Add H₂O to 500 ml.

Solution 2:	per 500 ml:
1% SDS	50 ml 10% SDS
0.2 N NaOH	100 ml 1 N NaOH

Add H₂O to 500 ml.

Solution 3:	per 500 ml:
3 M K+	300 ml 5 M Potassium Acetate
5 M Acetate	57.5 ml glacial acetic acid

Add H₂O to 500 ml.

TE	per 100 ml:
10 mM Tris-HCl pH 8.0	1 ml 1 M Tris-HCl pH 8.0
1 mM EDTA	0.5 ml 0.5 M EDTA pH 8.0

Add H₂O to 100 ml. Optional: RNAse can be added to TE at final concentration of 20 ug/ml.

 

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