SHEATECH, INC
Gene Expression and Proteomics

Mini-prep/Midi-prep

Protocol for Mini-prep


1. Pick a colony and grow in 3 mL of LB medium with antibiotic (type of antibiotic depends on    the plasmid) in shaker at 250 rpm, 37°C, overnight.
2. Pour approximately 1.4 mL of culture into microcentrifuge tube. Centrifuge at maximum speed for approximately 20-30 seconds. (Pellet will appear.)
3. Pour out the supernatant. Add 150 uL of solution 1. Vortex until pellet disappears and completely resuspends.
4. Add 150 uL of solution 2. Gently invert up and down ten times. (Solution will become viscuous.) Open the cap and allow the solution to settle at room temperature for 5 minutes.
5. Add 150 uL of solution 3. Gently invert up and down ten times. (Genomic DNA which is white in appearance will precipitate.) Do not vortex.
6. Centrifuge at maximum speed for 5 minutes.
7. In a new microcentrifuge tube, add 450 uL of isopropanol. Transfer the supernatant into the new microcentrifuge tube. Invert several times. Do not vortex.
8. Centrifuge at maximum speed for 5 minutes. (A clear/white pellet barely visible to the naked eye will form.)
9. Remove supernatant and place upside down to air dry the pellet on a clean paper towel.
10. Add 400 uL of 70% EtOH to wash the pellet. Invert up and down once or twice.
11. Centrifuge at maximum speed for 2-3 minutes. Repeat step 9.
12. If the pellet still is not dry, invert upside down to air dry at room temp for 10 minutes and place the microcentrifuge tube into the 37°C incubator for 20-25 minutes. Do not overdry the pellet.
13. Add 30 uL of 0.1xTE. Tap to mix. You may incubate the sample at 37°C while preparing restriction enzyme digestion.







Protocol for Midi-prep



1. Pick a colony and grow in 3 mL of LB medium with antibiotic (type of antibiotic depends on the plamid) at 37°C, 250 rpm overnight in a 15 mL sterilized tube.
2. Add 35 mL of amp LB medium into an Erlenmeyer flask. Transfer 300 uL of the above into the flask. Again, grow overnight at 37°C, 250 rpm.
3. Transfer to a 50 mL centrifuge tube. Centrifuge at 5000 rpm for 5 minutes using the eppendorf 5804.
4. Remove the supernatant. Do not disturb the pellet. Add 2 mL of Solution 1. Vortex to resuspend the pellet.
5. Add 2 mL of Solution 2. Do not vortex, simply invert up and down ten times. (Solution will become viscuous.) Open the cap and allow the mixture to settle at room temperature for 5 minutes.
6. Add 2 mL of Solution 3. Do not vortex. Invert and down ten times. (Genomic DNA which is white in appearance will precipitate.)
7. Centrifuge at 9600 rpm for 5 minutes using eppendorf 5804.
8. Spin column preparation:
    a. Remove the blue cap and white stopper.
    b. Allow solution to flow through via gravity.
    c. Transfer 5 mL of Solution A to the spin column.
9.    After centrifugation, transfer the supernatant to the spin column. Allow the solution to flow through via gravity; the DNA will be collected in the column.
10. Transfer 7 mL of Solution B to the spin column.
11. Blot the tip dry with a kim-wipe.
12. Obtain a new 5 mL sterile tube and attach it to the spin column with tape. Transfer 1.8 mL of Solution C (Elution buffer) to the spin column. The DNA will elute out along with the solution.
13. Transfer .9 mL of the solution from the sterile tube to a microcentrifuge tube containing 600 uL of isopropanol. Repeat this step another time.
14. Gently invert both tubes up and down.
15. Centrifuge at maximum speed for 10 minutes.
16. Remove supernatant and place upside down to air dry the pellet on paper towel.
17. Add 400 uL of EtOH to wash the pellet. Shake up and down once or twice.
18. Centrifuge at max speed for 2-3 minutes. Repeat step 16.
19. If the pellet still is not dry, place the microcentrifuge tube into the 37°C incubator for 5-10 minutes. Do not overdry the pellet. Be sure to check periodically.
20. Add 75 uL of 0.1xTE to each microtube.

To  measure the amount of DNA present using a spectrophotometer set at OD 260.
    a. Change the wavelength to 260 nm.
    b. Zero base using 800 uL of the appropriate solution.
    c. Add 4 uL of DNA mixture and 796 of the above solution to a new microtube. Mix well and transfer to cuvet.