Introduction
Extraction and purification of plasmid DNA from E.coli can be performed on a small scale,where DNA is extracted from just 1-2 ml of bacterial culture (mini-prep), or from much larger volumes (100 ml Ð maxi-prep; 1 L Ð giga-prep). The procedure requires cell lysis to release DNA (and other cell components including RNA and protein) and then DNA purification. The procedure involves two steps performed over two days:
1st day Ð Inoculation and growth of the bacteria that contain the plasmid Bacteria are grown in nutrient medium supplemented with antibiotics for 12-16 hours (usually overnight) to amplify the plasmid.
2nd day Ð DNA extraction and purification
The bacterial cell wall is disrupted by Òalkali-lysisÓ to release DNA which is then separated from the rest of the cellular components. Traditional methods have included the use of organic solvent extractions (e.g. phenol extraction was used for mini-preps) or ultracentrifugation (e.g. CsCl gradient ultracentrifugation for maxi-preps). The most common method these days is the use of commercially available column (see section 3.2.1).
Aims
During this workshop, you will perform mini-preps twice. We will assess DNA purity and yield by gel electrophoresis, although details are given for measuring DNA spectrophotometrically. The appendix lists alternative methods and larger scale purification protocols.
Time scale
Mini-preps involve an overnight growth step:
Day 1(PM) Set up mini-prep cultures from the TOPO-cloning expt Ð details of which colonies to inoculate will be decided on the day.
Day 2(AM) Perform mini-preps
1st Day Ð culture inoculation and growth
Materials required
Sterile L-broth media supplemented with appropriate antibiotic (e.g. ampicillin @ 100 µg/ml)
Wire loop Bunsen burner
Culture inoculation
1. Assess the success of the cloning reaction, count and record the number of colonies on each plate.
2. Inoculate 2 ml of L-broth containing appropriate antibiotic with a single colony from the appropriate plate using a sterile yellow pipette tip or a sterilised wire loop.
3. Incubate the culture(s) at 37℃ overnight with shaking (shaking is essential to aerate the culture and thereby ensure maximal growth and plasmid DNA yields).
2nd Day -DNA extraction and purification
Materials required
QIAGEN Mini-prep kit Microfuge
Cell Lysis/DNA Extraction
1. Concentrate the bacteria by transferring 1 ml bacterial suspension to an Eppendorf tube and microfuge at 12,000 rpm for 1 min.
2. Resuspend the bacterial pellet in 250 µl of Buffer P1.
3. Add 250 µl of Buffer P2, mix gently, and incubate at room temperature for 5 min.
4. Add 350 µl of N3, mix thoroughly but gently by inverting 4-6 times.
5. Centrifuge at 13,000 rpm in a microfuge for 10 min. Remove supernatant promptly. (During centrifugation, place a QIAprep spin column in a 2 ml collection tube.
DNA Purification
1. Apply the supernatants from above (step 5) to a QIAprep column by decanting.
2. Centrifuge for 1 min at 13,000 rpm. Discard the flow-through.
3. Wash the QIAprep spin column by adding 500 µl of Buffer PB and centrifuging for 1 min at 13,000 rpm. Discard the flow-through.
4. Wash QIAprep spin column by adding 750 µl of Buffer PE and centrifuging for 1 min at 13,000 rpm. Discard the flow-through.
5. Centrifuge for an additional 1 min to remove residual wash buffer. Place QIAprep column in a clean 1.5 ml Eppendorf tube. To elute DNA, add 50 µl Buffer EB to the centre of each QIAprep column, let stand for 1 min, and centrifuge for 1 min.
6. Transfer the eluate to a clean labelled eppendorf tube-store on ice or at -20℃.
Appendix
DNA concentration measurement
DNA purity, integrity and concentration can be assessed by gel electrophoresis and spectrophotometry. For mini-prep DNA, gel electrophoresis is usually sufficient. For maxipreps,DNA is measured in a spectrohotometer at 260 and 280 nm. A ratio of 260/280 of 1.8-2.0 indicates purity
1. Dilute 1-10 µl of DNA into 500 µl H2O.
2. Place in a quartz cuvette and measure the OD260 and OD280.
3. Calculate the DNA concentration using the following equation:
