Thermo Fisher Scientific Prod Single-strand Cir DNA Molecules Supercoiled Dble-strand Plasmids User guide

Brand
Thermo Fisher Scientific
Model
Prod Single-strand Cir DNA Molecules Supercoiled Dble-strand Plasmids
Type
User guide
Protocol
Production of Single-stranded Circular
DNA Molecules from Supercoiled
Double-stranded Plasmids in vitro
1. Add the following components to a reaction tube:
2. Vortex the tube and spin in a microcentrifuge
for 3-5 seconds.
3. Incubate at 37 °C for 1 hour.
4. Add 0.5 S volume of phenol (200 μL) and
0.5 S volume of chloroform/isoamyl alcohol
(24:1) (200 μL), vortex for 10 seconds and
centrifuge at 10,000 rpm for 5 minutes.
5. Transfer the upper aqueous phase to a fresh tube
and add 1 volume (400 μL) of chloroform/isoamyl
alcohol (24:1). Vortex and centrifuge for 5 minutes.
6. Repeat Step 5 twice more.
7. Transfer the upper aqueous phase to a fresh tube.
Add 1/10 volume of 3 M sodium acetate and
2.5 volumes of ice-cold ethanol. Mix and
incubate at -20 °C for 1 hour.
8. Centrifuge at 10,000 rpm for 10 minutes.
9. Pour off the supernatant and carefully wash
the pellet with 200 μL of 75% ice-cold ethanol.
Dry the pellet.
10. Dissolve DNA in 50 μL of Water, nuclease-free.
11. Treat with Thermo Scientic Exonuclease III
(Cat #EN0191) by adding the following
components:
12. Mix and incubate at 30 °C for 10 minutes. Stop
the reaction by heating at 70 °C for 10 minutes.
13. Extract the reaction mixture with phenol/
chloroform as described in steps 4-6, precipitate
DNA as described in steps 7-9 and dissolve in
10-30 μL of deionized water. This solution should
contain single-stranded DNA, suitable for DNA
sequencing, site-specic mutagenesis, differential
display, etc.
This protocol is for the Production of Single-stranded Circular DNA Molecules from Supercoiled
Double-stranded Plasmids in vitro.
Plasmid containing
Bpu10I recognition
sequence (20 µg)
20-356 μL
10x Buffer R 40 μL
Thermo Scientic
Nb.Bpu10I (Cat #ER1681) 4 μL (20 U)
Water, nuclease-free to 400 μL
10x reaction buffer
for ExoIII 25 μL
Exonuclease III 6 μL (1200 U)
Water, nuclease-free 169 μL
thermoscientific.com/onebio
© 2012 Thermo Fisher Scientic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries.
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Thermo Fisher Scientific Prod Single-strand Cir DNA Molecules Supercoiled Dble-strand Plasmids User guide

Brand
Thermo Fisher Scientific
Model
Prod Single-strand Cir DNA Molecules Supercoiled Dble-strand Plasmids
Type
User guide
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