Cleavage of F88-4 with Hindiii and Psti

0a9492ddfd9d91c879722c42675b0d3f.doc 04/06/18 Page 1

Cleavage of f88-4 with HindIII and PstI

The Type 88 vector f88-4 (see vectors.doc) has HindIII and PstI cloning sites separated by a 21- bp stuffer. Here is a protocol we recently used to cleave this RF. Note that we cleaved first with HindIII, then with PstI; that’s because PstI is reported to work well on sites that are close to ends, while HindIII is reported not to always work well on such sites. In this protocol, we use isopropanol precipitation rather than ultrafiltration through Centricon 100’s (see VectorCleavage.doc) to remove the stuffer.

1. Into a 15-ml screw-cap conical tube dilute 456 µg f88-4 RF (in TE or other non-interfering buffer) with sufficient water to bring the total volume to 2038 µl.

2. Into the 15-ml tube pipette

228 ml 10´ REact 2 buffer 1140 units (~14.25 ml) high-concentration HindIII

3. Incubate 2 hr 37º.

4. Add:

228 ml 10´ REact 2 buffer 2033 ml water 1140 units (~19 ml) high-concentration PstI

Total vol = 4.56 ml

5. Incubate 2 hr 37º.

6. Extract once with 4.5 ml neutralized phenol and once with 4.5 ml chloroform, using the double-spin method.

7. Transfer the final aqueous phase into a tared 30-ml glass Corex tube. Note net weight. Add TE to bring the total net weight to 12.31 g.

8. Add 1.368 ml 3 M NaOAc pH 6; vortex; add 6.44 grams (= 8.208 ml) isopropanol; cover with parafilm and vortex; incubate on ice 30 min.

9. Centrifuge 30 min 12,000 rpm at 4º in the SS34 rotor (use thin-walled rubber adaptor and appropriate balance tube), marking the centrifugal side of the tube so you will know where the invisible pellet is; RRR; wash with 20 ml freezer cold 70% ethanol; RRR; dry briefly under vacuum.

10. Dissolve the pellet in 900 ml TE by scraping and pumping up and down along the centrifugal wall of the tube; spin briefly in the clinical centrifuge to drive the solution to the bottom; transfer evenly into two 1.5-ml Ep tubes (450 ml each); to each tube add: 0a9492ddfd9d91c879722c42675b0d3f.doc 04/06/18 Page 2

45 ml 3 M NaOAc pH 6 1 ml ethanol

Vortex; incubate on ice at least 1 hr.

11. Microfuge in cold 30 min; RRR; wash with ~1 ml 70% ethanol; RRR; dry pellets briefly in SpeedVac. Dissolve each pellet in 375 ml TE, and pool both pellets in a single one of the Ep tubes.

12. Scan 200 ml of a 1/50 dilution from 220-300 nm. Last time we did this, we got an undiluted concentration of 342.5 µg/ml, corresponding to 257 µg altogether, or 56.3%.