Keating:Experimental Protocols:Capillary Electrophoresis

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Instructions for using Beckman PACE MDQ capillary electrophoresis

 * written by Nora 4/21/05


 * 1) Prepare buffers and samples
 * 2) *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)
 * 3) *when in doubt, start with borate buffer pH 8.0
 * 4) *concentration 20-200mM (start with 50mM)
 * 5) *salt in buffer will interfere with conductivity and separation
 * 6) *need 3 vials of each buffer (~1.5ml each) – top with orange caps
 * 7) *don't fill past the shoulder of the vial
 * 8) *samples should be approximately 50uM in a minimum volume of 50ul of sample buffer (same as running buffer)
 * 9) *not much of the sample will be injected and the majority of the sample can be recovered at the end of the experiment
 * 10) *place samples in PCR tubes in plastic vials with springs, topped with gray caps
 * 11) *also prepare water, 0.1M HCl, 0.1M NaOH, methanol, and waste vials
 * 12) *filter all samples and buffers with 0.22um filters
 * 13) *make fresh buffer aliquots for every ~10-20 runs / 2-3 weeks
 * 14) Open PACE MDQ software program - 32Karat
 * 15) *software is very similar to the HPLC software
 * 16) *instrument should already be on
 * 17) Turn on UV lamp to warm up for at least 15 min
 * 18) Make a directory for yourself in D:\Users\CE
 * 19) Sign in the log book
 * 20) Change cartridge and/or capillary if desired.
 * 21) *in software, manual control – press load to move vial trays forward
 * 22) *open tray and cartridge cover
 * 23) *unscrew the insertion bar keeping the cartridge in place and remove the cartridge
 * 24) *follow instructions in the installation and maintenance manual to change the capillary
 * 25) *reverse instructions to replace
 * 26) Place sample and buffer vials in buffer and sample trays
 * 27) *pay attention to where the tray positions for writing the methods
 * 28) *below is the standard positions for the minimum buffers needed
 * 29) *samples placed in the back sample trays can be kept at particular temperature, but can also be placed in buffer trays (only RT)
 * 30) Methods
 * 31) *use examples in D:\Users\CE\General Methods\
 * 32) *if it's the first time using the capillary ever or in several months, start with condition.met capillary to go through extensive rinsing
 * 33) *otherwise use wash.met to do a quick rinse
 * 34) *for separation methods, use bufferXaYb.met where Xa is position of rinse buffer vial and Yb is position of separation buffer vial
 * 35) *separation methods include a few washes, injection of the sample, and separatation of the sample under voltage
 * 36) *method files assume injection of buffer as the sample (baseline)
 * 37) *after all runs have been performed, use shutdown.met to give the capillary a final rinse, includes turning off the lamp
 * 38) *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
 * 39) *to run a single method, press the blue arrow
 * 40) *save the data file in your directory's data folder
 * 41) Sequences
 * 42) *to run multiple samples, use a sequence file to specify the method, data file, and sample position of each sample
 * 43) *for examples, look in D:\Users\CE\Nora\Sequences
 * 44) *you need to name the data files by typing in the sample, including the entire path with your directory
 * 45) *press the green arrow to start the sequence
 * 46) *sequence can be modified in the later steps and saved while running
 * 47) Notes
 * 48) *if window pops that coolant is low, refill coolant
 * 49) **open bottom panel
 * 50) **connect tube attached to syringe
 * 51) **pour in coolant in 5ml increments, just let flow in
 * 52) **until level of coolant in viewing tube is between the black lines