Sequencing problem

A.J.Cann nna at
Sat Jun 10 05:59:35 EST 1995

wyu at (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:
 6.5µl water
 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)
 Total: 8.05µl

 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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