Preparing Phage to Measure Dipolar Coupling Constants

 

·       You need to use the Optima Max ultracentrifuge at Room 250 in the biology building.

·       Rotor to use MLA-130- Beckman. Put the rotor in the refrigerator 30 minutes in advance (on top of a kimwipe). The rotor has to be on a kim wipe at all times and never on a bare counter.

·       Phage to be used:   Pf1-LP11-92   (from ASLA)

Magnetic resonance cosolute

#P100-RNA

      Kept in storage buffer: 10mM sodium phosphate buffer

                                            pH 7.6 in DEPC treated water

      Phage stock concentration : ~50 mg/ml

      For each partially labeled RNA sample, you need approximately 16-17mg/ml of final

      phage concentration.

      Estimate approximately 20% losses and pipet out phage accordingly into the quick seal

      centrifuge tubes.

·       Rinse the centrifuge tubes in advance with Rnase Zap, water and autoclave before use. Also, make sure that the tubes are dry before use. Wipe pipets with Rnase Zap too.

·       The phage solution is viscous, so make sure you pipet slowly and correctly from the stock solution. You could use a sterile syringe to remove the bubbles. Rinse the pipet tip with buffer and fill up the centrifuge tube to a volume of 2 ml. There is a mark on the tube to indicate the volume. It is easiest to use a syringe for this step.

·       Buffer :     10mM sodium phosphate buffer in D2O

5mM magnesium chloride

pH 8.0

      Note: Keep buffer and phage on ice at all times!!

·       Turn on the centrifuge in the meantime and set temperature to 40C.

·       Seal the tube on top with the sealing iron instrument and make sure it is not leaking using a kim wipe. Weigh the tube and make a balance with a similar tube. Since the phage is denser than water, you may need to dilute glycerol in water to make the balance. Be as accurate as possible. * Remember to label the balance and the tube.

·       Put the rotor into the centrifuge, balance the tubes in the rotor. Put the floating spacers on top of the tube.

Set up the centrifuge at: 90000rpm for 1 hour at 40C. Make sure the centrifuge goes up to maximum speed before leaving the place and make sure that you have signed into the log book.

 

 

 

 

 

 

 

·       After centrifugation, you see a clear glassy pellet in the tube. Use a Rnase zapped razor blade to cut the top of the tube. Dump out the liquid as long as you see a solid pellet. Remove the remaining liquid with a pipet.

·       Resuspend the pellet in buffer and transfer it to a new tube. Rinse the pipet tip and tube thoroughly with buffer, since the phage is sticky!! Fill up the tube again as before and repeat the spin. You should do this whole resuspension and centrifugation step atleast  four times with buffer following the same protocol. In case any of the tubes collapse in the centrifuge, make sure you clean all the liquid in the rotor before starting the next round.

·       We decided to resuspend the pellet after four washes in deuterated tris (from powder: less chances of contamination than using opened liquid. In future, we could use deuterated tris in ampules.) Filter the 1X NMR buffer through a 0.22 micron filter. Rinse the filter first with D2O to remove glycerol.

·       Make sure you clean up and put everything back to where it belongs before leaving their lab!!

·        Resuspend the pellet in 250µl of NMR buffer:

10mM deuterated tris

0.1mM EDTA

0.04% sodium azide

pH 8.0

·       Measure the phage concentration. Turn the UV on and measure the absorbance at 270 (close to the extinction coefficient of the phage). Repeat 2-3 times at different dilutions to confirm the concentration. You need a final phage concentration of about 34mg/ml.

 

·       Double check the phage concentration by NMR.

Transfer the resuspended phage to a shigemi tube, put the plunger insert and parafilm and quick spin to remove the bubbles.

 

·       How to obtaina  2H 1D spectra:

Turn the lock and sweep off on BSMS.

Use zg program

In EDASP:  F1               2H            FCU1              X300 W             X-BB19F-2HS

O1P = 4ppm

P1 = 20

PL1= 8dB

 

 

 

 

 

 

 

Hard wire changes:

Unplug the 2H transmitter cable from the rear of the pre amp box.

Also, in the front of the preamp box: Unplug the X-nucleus cable and plug in the 2H cable instead.

 

Also, deuterium splitting can be detected by looking at the lock.

Run zg with ns =1and measure the splitting with a cursor. Difference between the values gives the value of J.

 

**Relationship between splitting (Q) and concentration:

QCC(2H ) = 0.886 * Cpf1 mg/ml

C= concentration of the phage.

 

 (Reference:  M. Zweckstetter and A. Bax; JBNMR. 20, 365-377. 2001)

 

Double check the concentration by increasing the number of scans, increasing TD to 16K or decreasing SW to 4K. All these experiments should give you about the same phage concentration.

 

·       Put all the NMR wirings back to normal.

·       Add about 16mg/ml of partially or completely labeled RNA to the phage in the tube and mix it with a long tip glass pipette. Wash and bake the pipette before use.

·       Put in NMR and first check a quick 1D. This time , in EDASP, F2              C13.

·       Run a constant time HSQC and IPAP and make required changes to optimise for the aromatics and ribose as required for your sample. Confirm the values from the header file (edcpul).