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Cleavage of Bacteriophage Vector f88-4 with HindIII and PstI
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
The Type 88 vector f88-4 (see Protocol ID#2176) contains HindIII and PstI restriction enzyme cloning sites separated by 21 bp (the "stuffer"). This protocol describes cleavage of this RF with the two enzymes. This protocol is similar to Protocol ID #2171 and employs an isopropanol purification step to purify the digested vector from the stuffer fragment.
Procedure
1. In a 15 ml screw-cap conical centrifuge tube, add TE to dilute 456 μg of f88-4 Replicative Form (RF) DNA to a final volume of 2038 μl.
2. Add 228 μl of 10 X REact 2 Buffer.
3. Add 1140 Units (14.25 μl) of high concentration HindIII.
4. Incubate the tube at 37°C for 2 hr.
5. Add 228 μl of 10 X REact 2 Buffer after incubation.
6. Add 2033 μl of ddH2O.
7. Add 1140 Units (19 μl) of high-concentration PstI (see Hint #2).
8. Incubate the tube at 37°C for an additional 2 hr.
9. Extract the solution once with 4.5 ml of Neutralized Phenol using the double-spin method (see Hint #3) as follows: Add the phenol to the tube, vortex to mix, and centrifuge at 1,000 X g in a centrifuge for 5 min to separate the phases. Carefully remove the organic (lower) phase of the solution, leaving all of the interphase and aqueous (upper) phase in the tube (see Hint #4). Centrifuge the tube again to re-separate the phases. Carefully collect the aqueous phase into a new tube, avoiding any interphase or organic solution.
10. Extract the aqueous phase once with 4.5 ml of Chloroform as in Step #9.
11. Pre-weigh an empty 30 ml Corex tube. Transfer the final aqueous phase into the tube and note the net weight. Add TE to the tube until the solution weighs 12.31 grams.
12. Add 1.368 ml of 3 M Sodium Acetate and vortex. Add 6.44 g (8.208 ml) Isopropanol, cover with parafilm, and vortex.
13. Incubate on ice for 30 min.
14. Centrifuge in a Sorvall™ SS-34 rotor at 12,000 rpm (17,200 X g) for 30 min at 4°C. Mark the centrifugal side of the tube to identify the location of the invisible DNA pellet (see Hint #5).
15. Carefully aspirate the supernatant, avoiding the marked side of the tube. Add 20 ml of -20°C 70% (v/v) Ethanol and centrifuge, as in Step #14, for 5 min. Aspirate the supernatant and dry the DNA pellet briefly under vacuum.
16. Dissolve the pellet in 900 μl of TE with trituration along the wall of the tube marked by pen in Step #14.
17. Centrifuge the tube for 1 min at 700 X g in a clinical centrifuge and transfer the DNA solution equally into two 1.5 ml microcentrifuge tubes (450 μl each).
18. Add 45 μl of 3M Sodium Acetate and 1 ml of 100% (v/v) Ethanol to each tube. Vortex the tubes then incubate them on ice for at least 1 hr.
19. Centrifuge the tubes at maximum speed in a microcentrifuge at 4°C for 30 min.
20. Aspirate the supernatants and add 1 ml of 70% (v/v) Ethanol to each tube. Invert to mix and centrifuge for 5 min at maximum speed in a microcentrifuge. Aspirate the supernatants and dry the DNA pellets briefly under vacuum.
21. Dissolve each pellet in 375 μl of TE and pool both pellets in a single microcentrifuge tube.
22. Scan 200 μl of a 1:50 dilution of the DNA solution at an optical density range from 220 to 300 nm (see Protocol ID#2174). Expect a recovery of approximately 50 to 60%.
Solutions
REact 2 Buffer (10X)
500 mM NaCl
Autoclave to sterilize and store at room temperature
100 mM MgCl2
500 mM Tris-Cl, pH 8.0 
TE
10 mM Tris-Cl, pH 8.0
1 mM EDTA
70% (v/v) Ethanol
Sodium Acetate
Store at room temperature
Adjust pH to 6 with Glacial Acetic Acid
3 M Sodium Acetate
Autoclave in a tightly-stoppered screw-cap bottle to prevent evaporation of Acetic Acid
Chloroform with Isoamyl Alcohol
Swirl to mix and store at 4°C
(CAUTION! See Hint #1)
Add 20.8 ml of Isoamyl Alcohol to 500 ml of Chloroform in the original bottle containing Chloroform.
Neutralized Phenol
Allow phases to separate and remove the aqueous (upper) phase
Equilibrate with Tris once more
Use water-saturated Phenol
Add one-tenth volume of 1 M Tris-HCl, pH 8.0
Shake or vortex vigorously to equilibrate phases
(CAUTION! See Hint #1)
Use the lower phase as Neutralized Phenol
BioReagents and Chemicals
HindIII Restriction Enzyme, High-concentration
Sodium Chloride
Acetic Acid, Glacial
EDTA
Isopropanol
Tris
Magnesium Chloride
Isoamyl Alcohol
Chloroform
Sodium Acetate
Phenol
PstI Restriction Enzyme, High-concentration
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. Note that the DNA is cleaved first with HindIII followed by PstI because PstI is reported to work well on sites that are close to ends while HindIII is reported not to work well on such sites.
3. The contributor suggests the use of the double-centrifugation method in order to increase the yield of the aqueous phase from each organic extraction.
4. The purpose of removing the organic phase is to lower the interphase into the narrow tip of the microcentrifuge tube so that the aqueous phase can be drawn off with high yield. Avoid removing the aqueous phase.
5. Use thin-walled rubber adaptors and an appropriately balanced blank tube. Mark the tube with an alcohol-resistant marker.
