Keating:Experimental Protocols:Capillary Electrophoresis

From OpenWetWare
Jump to: navigation, search
Information concerning the
Keating Lab

Lab Members

Experimental Protocols
Coder's Corner

back to Experimental Protocols

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