GE 50 reactions 79760 Recording Equipment User Manual


 
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dilute—approximately 0.8X strength. Be certain to run glycerol tolerant gels at
the same power (wattage) as TBE-buffered gels so the gel temperature is
normal.
10X TBE Buffer (70454)
Tris base 108g
Boric acid 55g
Na
2
EDTA
.
2H
2
O 9.3g
H
2
O to 1000ml, filter (may be autoclaved)
This is the traditional sequencing gel buffer. It should NOT be used with the
polymerase supplied in this kit (Glycerol Tolerant Gel Buffer should be used).
Gel recipes (for 100ml of gel solution)
Standard gel
Gel conc. Acrylamide/ Urea 20X Gly. Tol. OR 10X TBE
(%) bis-acrylamide (7-8.3M) Gel Buffer Buffer H
2
O
6% 5.7g/0.3g 42-50g 5ml* - ~45ml
8% 7.6g/0.4g 42-50g 5ml* - ~45ml
6% 5.7g/0.3g 42-50g - 10ml ~40ml
8% 7.6g/0.4g 42-50g - 10ml ~40ml
Dissolve, adjust volume to 100ml with H
2
O, filter and de-gas. When ready to
pour, add 1ml of 10% ammonium persulfate and 25µl TEMED (N, N, N', N'-
tetramethylethylenediamine).
*Use 4ml for faster gel migration.
Formamide gel (for resolution of compressions)
Gel conc. Acrylamide/ Urea* 20X Gly. Tol. OR 10X TBE
(%) bis-acrylamide (7M) Gel Buffer Buffer Formamide H
2
O
6% 5.7g/0.3g 42g 5ml - 40ml ~10ml
8% 7.6g/0.4g 42g 5ml - 40ml ~10ml
6% 5.7g/0.3g 42g - 10ml 40ml ~5ml
8% 7.6g/0.4g 42g - 10ml 40ml ~5ml
*Warming to 35-45°C may be required to dissolve urea completely.
Adjust volume to 100ml with H
2
O, filter and de-gas. When ready to pour add
1ml of 10% ammonium persulfate and 100-150µl TEMED. The temperature of
the mixture should be 25-35°C—warmer mixtures will polymerize too fast while
mixtures below 20°C may precipitate urea. They will require higher running
voltage and run slower than urea-only gels. Prior to drying, these gels should be
soaked in 5% acetic acid, 20% methanol to prevent swelling. For more detailed
information, refer to TechTip #200 available from USB Technical Support or the
Technical Library at usbweb.com.