Imran Mungrue
imran.mungrue@utoronto.ca

RESUME

     METHODS

6) Manual Sequencing (Amersham/Pharmacia Kit)

* Dilute 2ug of each template to 32ul with water

* Add 8ul 2M NaOH

* Add 7ul 3M NaOAc (pH4.8) and 4ul water

* Add 120ul 100%EtOH, mix and place on dry ice for 15min

* Spin at full speed for 15min @ 4degC in microfuge

* Remove supernatant, and add 20ul ice cold 70% EtOH

* Spin for 10min, remove supernatant again

* Dry pellet at RT for 5min

* Re-dissolve in 10ul water

* Add
2ul primer (5-10 pmol)
2ul Annealing buffer

* Vortex, pop spin, incubate @ 65degC for 5min

* Incubate @ 37 degC for 10min

* Leave  at RT for 5min, pop spin

* Add 2.5ul Termination Mix to each tube labelled A C T and G

* Dilute enzyme (good for two templates)
1ul pol
4ul dilution buffer

* To template DNA + primer add the following and start reaction
3ul Labelling mix
1ul labelled dNTP (>10mCi/mL)
2ul diluted enzyme

Reaction proceeds as follows
0) DNA 1 Add labelling mix 1 at a time to each template
1) DNA 2
2) DNA 3
3) DNA 4
4) Warm termination solutions @ 37degC
5) DNA 1 Add 4.5ul to each stop tube (every 15sec.)
6) DNA 2
7) DNA 3
8) DNA 4
9)
10) DNA 1 Add 5ul Stop solution to each tube (every 15sec)
11) DNA 2
12) DNA 3
13) DNA 4

* Store samples at -20degC until use

* Denature @ 95degC for 5min prior to running