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

EDTA.2H

2

O 9.3g

2

 

 

H2O 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

H2O

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 H2O, filter and de-gas. When ready to pour, add 1ml of 10% ammonium persulfate and 25∝l 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

H2O

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 H2O, filter and de-gas. When ready to pour add 1ml of 10% ammonium persulfate and 100-150∝l 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.

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