GE 100 reactions 79770 Benutzerhandbuch

19

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 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 25

µ

l TEMED (N, N, N', N'-

tetramethylethylenediamine).

*Use 4ml for faster gel migration.

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 100-150

µ

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.