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  3. Prod Single-strand Cir DNA Molecules Supercoiled Dble-strand Plasmids

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

  • Hello! I've analyzed the user guide for production of single-stranded circular DNA molecules using Thermo Scientific protocols. The document details the use of enzymes like Bpu10I and Exonuclease III, and includes steps for plasmid processing and DNA purification. I'm ready to assist with any specific questions you might have about performing the protocol.
  • What is the purpose of this protocol?
    What enzymes are used in this protocol?
    What are the final applications of the produced DNA?
    What is the incubation temperature for Bpu10I digestion?
    What is the incubation temperature and time for Exonuclease III?
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
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