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Instructions for using Beckman PACE MDQ capillary electrophoresis

• written by Nora 4/21/05

1. Prepare buffers and samples
   • choose a buffer or set of buffers to use as the running and sample buffers, ideally ~2 pH units above pI of protein (if known)
   • when in doubt, start with borate buffer pH 8.0
   • concentration 20-200mM (start with 50mM)
   • salt in buffer will interfere with conductivity and separation
   • need 3 vials of each buffer (~1.5ml each) – top with orange caps
   • don't fill past the shoulder of the vial
   • samples should be approximately 50uM in a minimum volume of 50ul of sample buffer (same as running buffer)
   • not much of the sample will be injected and the majority of the sample can be recovered at the end of the experiment
   • place samples in PCR tubes in plastic vials with springs, topped with gray caps
   • also prepare water, 0.1M HCl, 0.1M NaOH, methanol, and waste vials
   • filter all samples and buffers with 0.22um filters
   • make fresh buffer aliquots for every ~10-20 runs / 2-3 weeks
2. Open PACE MDQ software program - 32Karat
   • software is very similar to the HPLC software
   • instrument should already be on
3. Turn on UV lamp to warm up for at least 15 min
4. Make a directory for yourself in D:\Users\CE
5. Sign in the log book
6. Change cartridge and/or capillary if desired.
   • in software, manual control – press load to move vial trays forward
   • open tray and cartridge cover
   • unscrew the insertion bar keeping the cartridge in place and remove the cartridge
   • follow instructions in the installation and maintenance manual to change the capillary
   • reverse instructions to replace
7. Place sample and buffer vials in buffer and sample trays
   • pay attention to where the tray positions for writing the methods
   • below is the standard positions for the minimum buffers needed
   • samples placed in the back sample trays can be kept at particular temperature, but can also be placed in buffer trays (only RT)
8. Methods
   • use examples in D:\Users\CE\General Methods\
   • if it's the first time using the capillary ever or in several months, start with condition.met capillary to go through extensive rinsing
   • otherwise use wash.met to do a quick rinse
   • for separation methods, use bufferXaYb.met where Xa is position of rinse buffer vial and Yb is position of separation buffer vial
   • separation methods include a few washes, injection of the sample, and separatation of the sample under voltage
   • method files assume injection of buffer as the sample (baseline)
   • after all runs have been performed, use shutdown.met to give the capillary a final rinse, includes turning off the lamp
   • any of methods can be modified, including absorbance wavelenth, sample/buffer positions, length of time for separation or washes, etc, but please save in your directory's methods folder
   • to run a single method, press the blue arrow
   • save the data file in your directory's data folder
9. Sequences
   • to run multiple samples, use a sequence file to specify the method, data file, and sample position of each sample
   • for examples, look in D:\Users\CE\Nora\Sequences
   • you need to name the data files by typing in the sample, including the entire path with your directory
   • press the green arrow to start the sequence
   • sequence can be modified in the later steps and saved while running
10. Notes
    • if window pops that coolant is low, refill coolant
      • open bottom panel
      • connect tube attached to syringe
      • pour in coolant in 5ml increments, just let flow in
      • until level of coolant in viewing tube is between the black lines