A. J. McKnight
26th April 2004
Preparation of WAVE Reagents
There are four reagents used during the running of the WAVE™. Before any
reagents can be made up, all glassware must be assembled and cleaned
thoroughly. Only dedicated glassware should be used for the WAVE™ and this is
located in the white cupboard below and to the left of the machine.
* All solutions should be prepared in the fume cupboard.
* Reagents A, B and C should be made up weekly.
* Monthly is sufficient for reagent D.
* All water used must be HPLC grade (18 mΩ ddH2O).
Materials required : 1 x 100 ml graduated measuring cylinder
2 x 2 litre bottles with screw-cap lids
2 x 1 litre bottles with screw-cap lids
2 x 250 ml beaker
3 sterile pastettes
1 x 100 ml Volumetric Flask
with appropriately
1 x 500 ml Volumetric Flask
sized stoppers
1 x 2 litre Volumetric Flask
All glassware must be washed by 1/4 filling each container with 18.2 mΩ ddH2O,
stoppering the container where appropriate and swirling the liquid vigorously.
Discard this water and repeat the procedure. As of 12/03/02, Transgenomic
recommend adding ~50 ml of ACN to each bottle (1 or 2 litre), swirling and
discarding the liquid. Then rinse bottle with copious amount of 18.2 mΩ dd
H2O. ***DO NOT AUTOCLAVE glassware***
3
/4 Fill bottles labelled A, B and D with mΩ ddH2O from the Millipore machine at the
rear of the lab.
♦ For optimum results from the WAVE™, all measurements must be
accurate. If any liquid is spilt during the transfer from one flask to
another, then the remainder should be discarded, appropriate glassware
washed and the procedure started again.
♦ Securely cap the bottles to prevent evaporation. Evaporation will lead to
an increase in ionic strength and volatilisation of the acetonitrile. Both
conditions will effect the performance of the instrument.
♦ On addition of water to ACN, the temperature of the buffer will drop – let
it come back to room temperature before adding the final amount of water.
♦ Remaining WAVE™ solutions should be disposed of into the designated
ACN waste bottles.
♦ No mineral oil, protein or air should be introduced to the column.
♦ Date each bottle containing new solution as it is made up – and append to
maintenance record. See Maintenance Records.
A. J. McKnight
26th April 2004
1.
i.
ii.
iii.
iv.
Buffer A - TEAA (pH7.0) is an ion-pairing reagent. TEAA is an ideal
medium for susceptible Stock in fridge is 2 M, but used at 0.1 M ⇒
dilute as follows:
Carefully pour 100 ml of TEAA from Transgenomic bottle into 100 ml volumetric
flask until the meniscus is resting on the marked line. Pour TEAA into the 2 litre
flask.
½ fill the small flask with mΩ H2O, swirl the contents and pour washings into the
1 litre flask. Repeat this last step three times.
Pour water from bottle A into the 2 l flask until it is ~ 2/3 full and mix the solutions
by inversion. Make the 2L flask up to the line with water from bottle A and again
mix by inversion.
Discard remaining water in Bottle A and transfer the buffer to bottle A. Seal
bottle A with screw cap lid and shake thoroughly.
100 ml TEAA + 900 ml water → Buffer A
2. Buffer B – 0.1 M TEAA as for A. 25 % ACN.
i.
ii.
iii.
iv.
Add 100 ml of TEAA to the 2 litre flask.
Pour 500 ml of ACN into the 500 ml volumetric flask and gently transfer this
ACN solution into the 2 l flask. Rinse the flask with mΩ dd H2O and add
washings to the 2 l flask.
Add water from bottle B to the flask until the flask is ~ 1/2 full, stopper the flask
and shake vigorously for several minutes. Top up the 2 l flask with water from
bottle B, making up to the line with water as before.
Discard remaining water in bottle B and carefully transfer the buffer from the
flask to bottle B. Cap the bottle and shake again to ensure thorough mixing of
reagents.
100 ml TEAA + 500 ml of Acetonitrile (ACN) + 1400 ml water → Buffer B
3. Solution C - 75 % ACN to clean the column ⇒ no known bacterial growth
possible
This solution uses more approximate measurements as it is used to wash the
WAVE™ and keep it clear of bacterial growth during periods of little use. This
solution is also used to clean the accelerator. Pour ~750 ml of ACN into Bottle C,
make up to 1l mark using mΩ H2O and mix.
750ml ACN + 250 ml ddH2O → 75 % ACN
4. Solution D / Syringe Wash - Wash / syringe solution to wash needle between
samples etc. 8% ACN.
Pour 80 ml of ACN from beaker into graduated measuring cylinder and transfer to 1 l
flask. Make up to 1 litre with water from bottle D and shake vigorously.
A. J. McKnight
26th April 2004
80 ml ACN + 920 ml ddH2O → 8 % syringe wash
" The new solutions should be placed beside those to be changed to the right of the
WAVE™.
*** Ensure that the tubing is not touched below the mark and that the inlet filter
is clear of obstructions during transfer. ***
" Filter tips should be placed in one hole of the buffer bottles.
" Potential introduction of air, during changeover to new buffers, should be negated
by purging for 2-5 minutes, or until no air can be seen in any of the solution lines.
See Purging buffer lines.