SEQUENCING GELS
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10X TBE, per liter Sequencing dye
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108g Tris base 80% formamide
55g boric acid 10mM NaOH
40ml 0.5M EDTA 1mM EDTA
0.1% xylene cyanol
10% APS 0.1% bromophenol blue
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dissolve 0.1g ammonium
persulfate in 10ml dH2O
and store frozen
GEL COMPOSITIONS
per liter 6% 8% 20%
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acrylamide 57g 76g 190g
bis acrylamide 3g 4g 10g
urea 500g 500g 500g
10X TBE 100ml 100ml 100ml
ddH2O 300ml 300ml 300ml
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Dissolve with mild heat, and adjust to 1 liter Filter, & store at room temperature in a closed container.
1. Clean 1 side each of 2 glass plates (one front plate, one back plate), first with ddH2O, then EtOH, then 3 times with acetone. Scrub hard & work a small area at a time on the second acetone wash. The cleaner the plates, the easier it is to pour a gel without bubbles.
2. Grease the corners of the bottom spacer & place it on the front plate. Grease the one end each of 2 side spacers & place them along the sides of the front plate, with the greased ends at the bottom. Fit the side spacers with the bottom spacer, & put extra grease at both spacer fittings. Lay the back plate onto the top plate, clean size down. Even-up the edges & clamp the edges together on the bottom & both sides with large clamps. Be sure the spacers are straight and fit together.
3. To 100ml of the appropriate gel mixture, add 50ul TEMED and 400ul 10% APS, then stir. Turn the plates over & prop up the front with an inverted 100ml plastic beaker. Tip one edge down and place your thumb down on that edge at the top, thus sealing off the plate overhang. Pour the acrylamide solution slowly into this corner next to your thumb, allowing the solution to flow down the edge into the plates. When the solution reaches the bottom, straighten the plates (i.e. no longer tilted from side-to-side) and allow the solution to fill the plates from the bottom up. Don't allow the solution above the smaller plate to all drain in - add more before this happens. If you don't, bubbles will be pulled into the gel. If a bubble is formed (or is about to be formed by uneven flow into the plates), tap the glass to dislodge it. If it won't come up, tilt the plates vertically, and bang the plates until the bubble is removed, then carefully lower the plates & continue pouring.
4. When the gel plates are full, pour more solution over the gel, then place the comb into the top of the plates through the crylamide solution (this prevents bubbles under the comb teeth). Clamp the comb into place & allow the gel to solidify - about 1 hour.
5. Remove the comb and the bottom spacer. Remove all but 2 clamps on each side, and clamp the gel into the gel box, being sure the corners at the top are sealed with plastic squares & vacuum grease (for our current units). Using a syringe & TBE buffer, rinse the air out of the wells and the bottom of the gel.
6. Heat the samples at 90C for 5 min, then cool on ice. While the samples are cooling, again rinse out the wells with buffer, being sure to rinse out all the urea-containing buffer from each well.
7. Using a drawn-out glass capillary & mouth pipetter, load each sample onto the gel. Run the gel at up to 80 watts, with the fan on.
8. When the gel is finished (depending on what you want to see, how big the DNA is, how many loadings you do, etc...), take the plates out of the gel box, and CAREFULLY separate the plates with a metal spatula by prying from one bottom corner. 6% gels can be lifted from the glass with a piece of 3MM paper rubbed over the gel - then covered with saran wrap & dried on a gel dryer. 8-20% gels are lifted using a used piece of X-ray film rubbed over the gel, then covered with saran wrap.
9. Autoradiography of hot samples is at room temperature; less hot samples are exposed with screens at -70C.
