1.GLYCOL/HEAT DENATURATION

DNA 7µl (3µg-5µg)
Plasmid Denaturing Reagent 5µl
Primer 1µl (
Mix thoroughly then incubate at 95°C for 5minutes

Chill on ice

Add Plasmid Reaction Buffer 2µl

2.ANNEALING

Incubate at 37°C for 10minutes, then chill on ice
-Aliquot 2.5µl of GATC Termination mix into vials
-Dilute Labeling mix 1:5 with water

3.PRE-WARM GATC Termination mixtures to 37°C

4.LABELING REACTION

To ice-cold annealed DNA mixture 15µl

add: DTT 1µl

Diluted Labeling Mix 2µl

35S dATP 0.5µl

Diluted Sequenase 2µl

Mix and incubate at room temperature for 5 to 10 minutes

5.TERMINATION REACTION

Transfer 4.5µl of labeling reaction into each tube, GATC

Incubate at 37°C for 5 minutes

6.STOP REACTION

Add 4µl of Stop Solution

7.HEAT SAMPLES

Heat samples to 75°C for 2 minutes immediately before loading

!!!!!!!!!!USE GLYCEROL TOLERANT GEL BUFFER ONLY!!!!!!!!!

8.MAKE GEL

Urea 25.2g
10x glycerol-tolerant gel buffer 7.2ml
Long Ranger acrylamide 6ml
Bring to 60ml with Milli-Q

This takes some time to go into solution

add 10% ammonium persulfate 300µl

TEMED 30µl