BISC411

                                              EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL

                                                             Mini-Prep Protocol

NOTE: This is the procedure that is included with the PerfectPrepTM Spin Mini Kit from the 5 Prime company.

1. You will be given 2 overnight cultures of bacteria containing 10 ml of a plasmid under the control of an inducible promoter. The two cultures will be different from one another. You will not know which is which. They will be marked as culture A, B, C, or D. Today you will purify the plasmids using an alkaline lysis procedure and a special DNA-binding column made by the 5 Prime company. Before beginning the purification, you will need to equilibrate the CB3 spin columns. You will be given 4 columns per group. To each, add 500 microliters of Buffer BL, place it into a collection tube, put into the minifuge, and spin for 1 minute. Discard the flowthrough. Put these columns aside for now.

2. We will pellet the bacteria out of their growth medium by centrifugation in a minifuge for 1 minute. Use only two 1.5 ml microfuge tubes per culture. Fill the tubes 3/4 of the way up, spin, decant and repeat until all of the volume has been centrifuged.

3. Decant the growth medium carefully into the bleach-containing bottles. After the last spin be sure to remove all the growth media possible using your pipetman tip. Then add 250 microliters of Buffer PR1 containing RNAase to each tube. This sets up the appropriate environment for successful lysis that will remove only the plasmid DNA in the bacteria without the chromosomal DNA.

4. Resuspend the pellets in this buffer. You may use the vortex to resuspend. DO NOT VORTEX AFTER THIS POINT IN THE PROTOCOL!

5. Lyse the bacteria by adding 250 microliters of Buffer PL2 to each tube . This is a NaOH solution that induces alkaline lysis of the cells, a procedure that does not release the chromosomal DNA but does allow the release of the plasmid DNA. Mix gently by inverting the tubes 4-6 times.

6. Neutralize the lysates by adding 350 microliters of  Buffer PN3 to each tube. Immediately mix again by inverting tubes 4-6 times. This buffer not only adjusts the pH back to neutral conditions but also provides a high-salt environment that will allow the DNA to be adsorbed on the purification column filter later.

7. Pellet the precipitated bacterial cell material by centrifugation for 10 minutes.

8. Carefully transfer the supernatants to clean 1.5 ml microfuge tubes and spin again for 5 minutes. Be sure to avoid transfering any particulate material.

9. Insert 4 CS columns into a new collection tube. Note that this is NOT the column you equilibrated in step 1, but the other column you were given. It is designed to help further remove contaminants. Carefully pipette your clarified bacterial lysates (superntant) into these CS columns. Be careful to note which lysates are from culture A and which from culture B.

10. Centrifuge the spin column for 2 minutes. DO NOT DISCARD THE FLOWTHROUGH.

11. Carefully apply the flowthrough from step 10, which contains your DNA,  into the equilibrated CB3 column (from step 1) in the collection tube used during the equilibration.

12. Centrifuge for 1 minute. Discard the flowthrough. Your DNA should be trapped on the column filter. The filter is a silica-based filter with high affinity for DNA that is in a high-salt buffer.

13. Add 500 microliters of Buffer PD to the column and centrifuge for 1 minute. Discard the flowthrough. This buffer removes endonucleases that could nick the DNA.

14. Add 700 microliters of Buffer PW to the column and centrifuge for 1 minute. Discard the flowthrough. This buffer removes salts and thus prepares the DNA for elution.

15. Without adding any more buffer, spin the columns again for 2 minutes to remove any residual buffer left on the filter.

16. Allow the column to thoroughly dry by leaving the cap open for 10 minutes. This is important. the wash buffer contain ethanol that must be vaporized before eluting the DNA.

17. Elute the DNA by putting the column into a clean 1.5 ml microfuge tube and adding 100 microliters of PEB buffer to the center of the column. This is a gentle Tris-EDTA buffer that is commonly used for storage of DNA. Incubate this for 5 minutes. Then centrifuge for 1 minute. The flowthrough is your eluted DNA. You should now have 2 eluates derived from culture A and 2 from culture B. Combine all the A eluates into one tube and all the B eluates into one tube. BE CAREFUL NOT TO MIX THE TWO TYPES.
 
18. Remove  25 microliters of your  DNA into a clean microfuge tube for use later in  measurement of your DNA yields.

19. To the remainder of your DNA samples, add one tenth volume of  5M NaCl (Since you have about 175 microliters remaining of your DNA samples after step 18, this would mean 17.5 microliters of the NaCl added. Then add 2.5 volumes of 95-100% ethanol. This would be 2.5 times 175 or 437.5 microliters of the ethanol. Mix well and put in the -20 degrees C freezer for 2 hours.

18. Come to the biophotometer with your 25 microliters of each DNA . I will measure your DNA concentrations by diluting your sample with an additional 25 microliters of TE and then reading the absorbance. (see step 22).

19. The biophotometer is set to read at wavelength 260.

20. The biophotometer is blanked using TE.

22. In a clean microfuge tube, 25 microliters of TE and 25 microliters of your DNA are carefully mixed, avoiding bubbles by using a p-20 and tip to do the mixing. The entire mixture is carefully added to a clean cuvette and placed into the biophotometer.

23. The absorbance is read directly from the biophotometer. It gives you the absorbance for that concentration of the DNA in the cuvette in micrograms per ml. Remember, the DNA has been diluted 1/2.

24. Calculate your actual DNA concentration for each DNA prep and your total yield of DNA from each bacterial culture.

32.  The biophotometer will also display the 280 reading.  A ratio of 260 reading to 280 reading that is no lower than 1.5  indicates "clean" DNA (little protein present). Record your ratios also to include in your lab report.

33. There is absorbance of ultraviolet light at 260 for DNA due to the presence of the nucleotide bases, which absorb maximally at this level. Protein will absorb mainly at 280, due to the presence of tryptophan and other aromatic side-chains in the amino acids. We should have little to no RNA contamination since our lysis buffers containe RNAase. If it is present, it would maximally absorb at 230. You do not need to take a 230 reading.

 34.  After the 2 hours of freezing are over, retrieve your ethanol precipitated DNA samples. Go to the cold room in room 116 McKinly and we will spin down the DNA by centrifugation at high speed for 10 minutes. When you get your spun samples back, look to see if you can see a tiny pellet in the bottom of the tube. That is your DNA. Sometimes it is too small to see.

35.  Decant the ethanol into a waste bottle and add 300 microliters of 70% ethanol as a wash. Spin again in the cold room for 4 minutes.

36.  Decant carefully, removing all of the wash bufffer but not throwing out the pellets!!.

37.  Determine how much TE you need to add to your pellet to make a 0.5 microgram per microliter concentration of your DNA. Add the appropriate amount to your tube and resuspend the pellet thoroughly in the TE. Store your DNA at -20oC. See below for a sample calculation.

38.  Note: For our future work, you will need at least 25 microliters of DNA. Therefore, even if your yield is low, resuspend in 25 microliters of TE. If you have a good yield of DNA that allows you to get the 0.5 micrograms per microliter concentration with more than 2 microliters, use that amount.

Sample calculation:  If your diluted DNA read 50 micrograms per ml on the biophotometer in step 23, that would mean your DNA is 50 times 2 or 100 micrograms per ml concentration, or .1 microgram per microliter concentration. Since you have 200 total microliters, your yield is .1 times 200 or 20 micrograms. Your pellet DNA contains 88% of your total yield or 17.5 micrograms of DNA. Therefore add 35 microliters of TE to each pellet and resuspend. You will now have 0.5 micrograms per microliter of your DNA.