C.B.S. Scientific SG Series - Manual (Page 20)

C.B.S. œ Scientific
20
Adjustable Height Sequencer
4.5 Recommended Buffers and Reagents
Pre-mixed acrylamide stock solutions are the method of choice. Use according to
manufacturer’s instructions.
EDTA solution
(0.5 M EDTA, pH 8.0, 100ml)
Na
2
EDTA•2H
2
O (FW 372.2) 0.5M 18.6g
Deionized H
2
O 70.0ml
Dissolve
10M NaOH. Adjust to pH 8.0.
∼5.0ml
Deionized H
2
O to 100.0ml
Store at room temperature
Tris-borate-EDTA buffer, 5X
(5X TBE, pH
∼8.3, 1 liter)
Tris (FW 121.1) 0.45M 54.5g
Boric acid (FW 61.83) 0.45M 27.8g
EDTA solution (0.5 M, pH 8.0) 0.001M 20.0ml
Deionized H
2
O to 1000.0ml
Do not adjust pH. Store at room temperature
Acrylamide stock solution
(40%, 19:1 acrylamide:bisacrylamide, 250ml)
Acrylamide
(FW 71.08) 95.0g
Bis* (FW 154.2) 5.0g
Deionized H
2
O to 250.0ml
Store at 4
°C protected from light.
*N,N’ Methylenebisacrylamide
Ammonium persulfate solution
(10% APS, Initiator, 1ml)
APS
(FW 228.2) 10% 0.1g
Deionized H
2
O to 1.0ml
Fresh APS “crackles” when water is added. If yours does not, replace it with
fresh stock. Prepare just prior to use.
Sequencing gel acrylamide solution
(6%, 100ml)
Acrylamide stock solution, 40% 6% 15ml
Urea (FW 60) 7M 42g
TBE, 5X 1X 20ml
Add deionized H
2
O to 100.0ml
Dissolve
Filter, dearate, and add:
APS, 10% solution 1ml
TEMED 60ul
Pour gel immediately.