For manual sequencing with T7 polymerase and 35S-labelled ddATP, apply step 0 - III.A. For "automatic" sequencing with Licor 4000L, in combination with cycle sequencing using Thermosequenase (Amersham), do step 0 - II and then III.B instead of III.A.
0. MEDIA AND REAGENTS
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Sarkosyl-mix: 0.4 % Sarkosyl
100 mM Tris pH 8
100 mM EDTA pH 8
Lysozym stock: 10 mg/ml (H2O), kept at -20oC.
Proteinase K stock: 10 mg/ml, kept at -20oC.
NaOH, HCl: 1N precisely.
LB + salts: LB + 1x basic M9-salts + 3% glycerol + antibiotics
(see protocol of ss-DNA prep.)
RNases: DNase free RNase A, RNaseT1. Stock 10 mg/ml (TE). Remove
DNase by heating for 15 min to 70oC and slowly cool down. Kept at -20oC.
PEG: 26.7% PEG 8000, 40 mM MgCl2)
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I. CULTURE
Make 1 ml liquid culture in Falcon snap cap tubes. Shake with maximum
inclination o.n. at 100 rpm, 37oC. Alternatively, scratch about
4 cm2 cell lawn from a plate, avoid agar pieces.
II. PLASMID EXTRACTION
1. Adjust heat block to 100oC or prepare boiling water bath.
2. Fill one eppy with culture, spin down for 3 min at max. speed. Remove
carefully and discard supernatant.
3. Add 600 ul Sarkosyl-mix, resuspend pellet with 200-Gilson pipette/yellow tip.
4. Add 25 ul lysozym stock, vortex briefly, incubate 10 min at rt.
5. Add 6 ul proteinase K stock, mix gently by inverting, incubate 1-2 hrs at
37oC, not longer!
6. Boil 40 secs in heat block (closed caps might pop, but no loss of material)
7. Spin 2 x 15 min max. speed at rt (turn tubes), remove and discard pellet
with tooth pick. No tube change. Yield: ca.400 ul.
8. Precipitate supernatant with 1 ml EtOH/AmAc for 15-30 min at 4oC,
wash with ethanol 80% and dry in heat block at 60oC.
9. Dissolve pellet in 20-100 ul TE. Yield is 2 - 30 ug.
Continue with step III or IV.
III.A. PRETREATMENT FOR MANUAL SEQUENCING :
(modified from K. Hsiao (1991). A fast and simple procedure for sequencing ds
DNA with Sequenase. NAR 19:2787.)
1. Set heat block to 75oC.
2. Take 5 ul plasmid DNA (1 ug)
3. Add 1 ul sequencing primer (Stocks: universal: 10 ng/ul; reverse, 50ng/ul)
4. Add 1 ul of 1N NaOH, incubate at 75oC for 5-10 min.
5. Add 1 ul of 1N HCl, mix.
6. Add 12 ul water
8. Precipiate with 50 ul EtOH/AmAc for at least 30 min at 4oC, wash with
ethanol 80% and dry in heat block at 60oC.
9. Dissolve pellet (template/primer) in 8 ul water
10. Add 2 ul 5xsequenase buffer
11. Anneal for >5 min at 37oC.
Continue with ss-DNA sequencing protocol, labelling step (III), but
add 1 u Klenow together with T7 polymerase.
III.B. PREATREATMENT FOR CYCLE SEQUENCING :
(modified from John Gardner, CEPRAP)
1. Take 50 ul plasmid DNA (~25 ug) in TE
2. Add RNAse A 50 ug/ml final concentration
3. Add RNAse T1 10 units/ml final concentration
4. incubate for 45-60 min at 37oC
5. precipitate with 125 ul EtOH/AmAc for at least 30 min at room temp.,
wash with ethanol 80% and dry briefly
6. Resuspend DNA in 50 ul TE
7. Add 250 ul water
8. Add 100 ul PEG solution
9. Let sit for 30 min at room temp.
10. Spin down for 8 min at room temp., max.speed
11. Resuspend DNA in 50 ul TE
12. Spin down undissolvable stuff for 5 min
13. Transfer supernatant (DNA) to new tube
14. Use 25 ul for cycle sequencing reaction
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