N. Benton 08-08-08
Reaction Setup:
1. Template amount: 13 µg for ~ 35kb genome
2. Primer amount: 20 pmol
3.
Customer should provide template and primer premixed in 7 µl total volume .
4. Make a master mix for the number of reactions being setting up plus 2 extra with the following per reaction amounts:
Big Dye Terminator v3.1 1.0 µl x (# of rxns +2)
5X Sequencing Buffer 2.0 µl x (# of rxns +2)
5M Betaine
(Sigma B0300-5VL)
1.0 µl x (# of rxns +2)
DIUF * check T/P volume, add water to bring it up to 7 µl.
5.
Add 4 µl of master mix to each well, then add the 7 µl of T/P mix for a total of 11 µl.
Cycling Parameters: 3700 w/ volume changed to 11 µl
96 o C for 2m
60 cycles:
96 o C for 30s
50 o C for 30s
60 o C for 4m
60 o C for 10m
4 o C hold forever
Reaction Cleanup:
SDS treatment and Edge DTR plates (see protocol)
-add 2ul of 1.1% SDS to reactions after cycling, mix
-heat for 5 min @ 98 o C
-cool to room temp for at least 10 min before loading samples onto the Edge
DTR plates