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3. For Seq Gel - want 125 ml/gel
IBI BRL
a. To 125 ml seq. gel mix, add: 80 ml add: 100 ml
468 ul AP, mix
140 ul Temed 300ul AP 375ul AP
89.6ul Temed 112ul Temed
b. Stir quickly with pipette
c. Pour to center of gel apparatus with electric pipetter. Fill to top.
d. Put on comb, flat side down, 1/811 BRL inside (between) 1/411 IBI glass plates. Clamp top with IBI
e. Lay f lat on 2 white test tube racks, and balance in center.
f. With black bulb, suck up rest of gel mix with pipette so you can see when your gel is hardened/solidifed.
4. Prepare lOx Buffer; pH it to 8.2 (see protocol) Prepare 1200ml lx Buffer and filter, sterilize itl
1000ml for BRL
5. Prep to run gel after solidified: ("Run to the red")
A. Remove bottom clips
B. Remove bottom spacer
C. Remove side clips
D. Carefully remove pop comb (lst squirt dH20 at top near and on combs to loosen it) - no bubbles (start at end and work your way up)!
E. Wash off top and glass with dH2o at sink, well
F. Fill heat black with dH20- Put this on a hot plate on lihigh".
G. Put gel and plates in apparatus and lock with 2 top locks only;
***or clamps at 2 top places with BRL right behind (1/211) spacer oarts *3 clamps each side).
H. Put lx TBE in top trough - check for leaks.
I. Put lx TBE in bottom trough.
J. Flush out area where comb was by pipetting in lx TBE from top trough.
K. Put in comb, teeth down, just enough to touch top of gel - make no ridges.
L. Mark gel glass plate as to how you will use it:
M. Use pipette to get rid of air bubbles between the glass plates at the bottom.
N. Check wells with seq. stop + dH20 to make sure that there are no leaks between wells, by adding a random amt. of stop + dH20 every other wells.
0. Run this into gel by pressing "power" and "start".
P. When heat block reaches 620C, put heat to "low", add any dH20 that might have evaporate to black = will be at 70 C for later use.
Load 2 sets of samples at a time by:
1. Using the exact pipetman, take 3ul of each and put in a new 72 well plate (previously labeled exactly as before).
2. Put 72 well plate on heat block 3 min at 700C. Get ice.
3. Clean out area f rom wells by f lushing w/ 1 x TBE, while samples are heating. FLUSH ALL WELLS.
4. Power "off"
5. Put 2.5 ul of each into seg. gel with 20 ul pipetman with yellow tips. Put in at an angle in back - so tip points down and toward you.
6. Power "on".
R. Heat another 2 sets (of 4) samples at 700C. Add 2.5 ul each
S. Repeat "RI' until all samples added. After start gelrecord watt and MAMP at beginning and end of gel run!
T. At 3 hrs, load short set = 2nd set of each sample (W should + 50). (3 hrs since this is when the BPB mix has reached the bottom of the gel)
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