nna at le.ac.uk
Sat Jun 10 05:59:35 EST 1995
wyu at uoguelph.ca (Wenjin Yu) wrote:
>I want to sequence a cDNA cloned in lambda gt11 directly. Although it is
>said possible I have never fond a reference. I really appreciate if your
>can provide me some information on this kind of work, for example how
>much DNA we should use for each sequencing reaction and the reference paper.
Double-Stranded Sequencing (Using Sequenase Kit)
1) Denature template:
9µl plasmid DNA (CsCl purified is best) ~9mg (=200ng/ml)
1µl 2M NaOH (=200mM)
Incubate 15min @ 37oC.
2) Add: 1µl 10mM primer (~66ng 20mer), 3µl 3M KAc, 75µl EtOH.
Chill on dry ice (5min), spin down in microfuge (5min). Wash pellet with 70% EtOH. Dry.
3) Resuspend in 2µl 5X Sequenase Buffer + 8ml water.
Dispense 2µl into 4 eppendorf tubes / microtitre wells.
4) Make Labelling Mix: / Template:
0.4µl 0.1M DTT
0.5µl 35SdATP (5µCi)
0.4µl Labelling Mix (undiluted, from kit)
(Dilute 10X to read sequences close to primer)
0.25µl Sequenase (undiluted)
Dispense 2µl per tube/well. Incubate 1-10min @ room temperature.
5) Add 2µl of the appropriate Termination Mix (from kit) to each tube/well.
Incubate 5min @ 37oC.
6) Add 4µl formamide/dye mix ("Stop Solution") per tube/well.
Heat to 80oC for 2 min, quench on ice.
Load 2-5µl (total 10µl) on gel.
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