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B. Sample Preparation
8
a. Speed vac samples down to dryness (here use 25ug/sample)
Store on ice until ready for next step.
b. Sample buffer:
Amt
Workin@ Stock Dil 1 samp 20samp 30 samp
Forraamide 50% 100% 1:2 15ul 300ul 450ul
Formaldehyde 12.3M 2.2M 1:559 lul 20ul 30ul
Running Buffer lx 20x 1:20 1.5ul 30ul 45ul
H20 12.5ul 250ul 375ul
30ul *600ul
our stock sample buffer solution
c. Resuspend each sample in 25 ul stock sample prep by pipetting
9. Prepare 2000 ml of Ix MOPS Running Buffer = lx RB; dilute 2ox to lx using inversion method to mix; use 100 ml 2OX MOPS, mark up to 2000 ml with dH20-
10. Put samples in 60-700C H20 bath in a holder for 5 min. Then, cool immediately on ice.
11.
a. Carefully remove tape from gel.
b. Fill electrophoresis apparatus with lx RB
c. Place black paper underneath
d. Put in gel (making sure RB is above gel)
e. Carefully remove comb
f. *Optional Check all wells with lx dye (in H20) to make sure they are intact before you load the samples. Be sure to run dye into the wells before adding the samples. Dilute sample buffer 1:10, then use well.
C. Sample Loading
12. Add 3ul sample buffer (glycerol/bromophenol blue) (1/10 vol.) to each sample at the edge of the eppendorf tubes
13. Shake rack so sample buffer goes down into sample.
14. Vortex samples; centrifuge quick spin.
15. Load samples in gel (33ul/sample)
16. Run gel (- to +red probe) at 50 V until all sample goes into well (approximately 15 min)
17. Hook up circulator and run at 20V overnight
You want to accomplish 350-400 volt hrs running a gel, 350 volt hrs divided by 16 hrs = 22 volts; 50 V for 15 min, 20 V overnight
Do not hook up circulator until samples are in gel (out of wells)
Sample Buffer: 50% glycerol
lmM EDTA
0.4% bromopheol blue
0.4% xylene cyanol
D. Blotting
1. Photograph gel
2. Soak gel in 5OmM NAOH for 20 min lOmM NaCl
3. Rinse gel in dH20
4. Soak gel in O.lM Tris pH 7.5 2x 201 each
5. Soak gel in lox SSC
6. Prepare gel for blotting as described in DNA protocol
7. Transfer buffer - lox SSC Nytran filter
8. Blot Overnight
9. Take down blot, soak Nytran in 5x SSC 51 at RT
10. Air dry blot
11. Bake blot 2 hours at 80 C
12. Photograph blot on UV box using rulers
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