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KLAUS DEICHMANN
5. Centrifuge for 10 min at 5°C (Eppendorf microcentrifuge).
6. Discard supernatant, wash pellet once with ethanol (70%) at -20°C.
7. Dry pellet briefly under vacuum, resuspend in 7 111 water.
Annealing
2. In a 1.7 ml microcentrifuge tube, combine
- 7 111 template solution
- 1 111 primer solution
- 2 111 annealing buffer
3. Using denatured plasmid DNA as template, incubate at 37°C for at least
15 min; using M13 DNA, heat to 65°C for 2 min.
4. Cool tube to room temperature over at least 30 min.
5. Meanwhile:
- Dilute the 5 xlabeling mix 5-fold with distilled water.
Dilute Sequenase™ to a concentration of 1.5 U/!11 using the appropriate dilution buffer supplied with the enzyme, keep on ice!.
Thaw labe!, dNTP-solutions (labeling-mix) and termination-mixes
and place on ice.
Label4 microcentrifuge tubes "A", "C", "G", "T".
Pipette 2.5 111 of the corresponding termination mixes into each of the
tubes; close lids and incubate at 37°C in a water bath.
Labeling
6. To the annealed template-primer-mix add:
- 1 111 DTT 100 mM
- 2 111 diluted labeling-mix
- 0.5 111 a[ 35 S]dATP
- 2 111 diluted Sequenase™
giving a total volume of 15.5 111. Mix carefully and incubate at room
temperature for 5 min.
Termination
7. Transfer 3.5 111 of the labeling reaction into each of the tubes containing
the termination mixes (use fresh tips and avoid contamination), spin 1
sec to mix and incubate for exactly 5 min at 37°C.
8. Add 4 111 stop solution to each of the tubes and spin for 1 sec to mix.
9. Pipette 3 111 of each of the reaction products into separate microcentrifuge tubes. Close lids firmly and heat to 75-80°C. Spin to collect the mixture at the bottarn of the tube, and keep on ice until ready to load the gel.
(Reactions can be stored for later use at -20°C).
KLAUS DEICHMANN
5. Centrifuge for 10 min at 5°C (Eppendorf microcentrifuge).
6. Discard supernatant, wash pellet once with ethanol (70%) at -20°C.
7. Dry pellet briefly under vacuum, resuspend in 7 111 water.
Annealing
2. In a 1.7 ml microcentrifuge tube, combine
- 7 111 template solution
- 1 111 primer solution
- 2 111 annealing buffer
3. Using denatured plasmid DNA as template, incubate at 37°C for at least
15 min; using M13 DNA, heat to 65°C for 2 min.
4. Cool tube to room temperature over at least 30 min.
5. Meanwhile:
- Dilute the 5 xlabeling mix 5-fold with distilled water.
Dilute Sequenase™ to a concentration of 1.5 U/!11 using the appropriate dilution buffer supplied with the enzyme, keep on ice!.
Thaw labe!, dNTP-solutions (labeling-mix) and termination-mixes
and place on ice.
Label4 microcentrifuge tubes "A", "C", "G", "T".
Pipette 2.5 111 of the corresponding termination mixes into each of the
tubes; close lids and incubate at 37°C in a water bath.
Labeling
6. To the annealed template-primer-mix add:
- 1 111 DTT 100 mM
- 2 111 diluted labeling-mix
- 0.5 111 a[ 35 S]dATP
- 2 111 diluted Sequenase™
giving a total volume of 15.5 111. Mix carefully and incubate at room
temperature for 5 min.
Termination
7. Transfer 3.5 111 of the labeling reaction into each of the tubes containing
the termination mixes (use fresh tips and avoid contamination), spin 1
sec to mix and incubate for exactly 5 min at 37°C.
8. Add 4 111 stop solution to each of the tubes and spin for 1 sec to mix.
9. Pipette 3 111 of each of the reaction products into separate microcentrifuge tubes. Close lids firmly and heat to 75-80°C. Spin to collect the mixture at the bottarn of the tube, and keep on ice until ready to load the gel.
(Reactions can be stored for later use at -20°C).