Preparation of Genomic DNA from Bacteria

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Purpose

This protocol can be followed to isolate DNA from a given bacteria for further use.

This protocol is based off the protocol found on pages 2.4.1-2.4.2 of Current Protocols in Molecular Biology

Materials Needed

TE Buffer

10% sodium dodecyl sulfate

20 mg/mL proteinase K

5 mL NaCl

CTAB/NaCl solution

24:1 chloroform/isoamyl alcohol

25:24:1 phenol/chloroform/isoamyl alcohol

Isopropanol

70% ethanol

Protocol

-Inoculate 5 mL of the appropriate broth for your culture and grow it at the appropriate conditions until the culture is saturated

-Spin 1.5 mL of culture at full speed in a microcentrifuge for 2 minutes and discard the supernatant

-Resuspend the cells in 567 microliters of TE buffer using a micropipet

-Add 30 micro liters of 10% SDS and 3 microliters 20 mg/mL proteinase K, mix well

-Incubate at 37 C for one hour

-Add 100 microliters of 5 M NaCL and mix

-add 80 microliters of CTAB/NaCl solution and mix

-Incubate at 65 C for 10 minutes

-Add an equal volume of chloroform/isoamyl alcohol

-Mix well and centrifuge at full speed for 5 minutes

-Transfer the aqueous supernatant to a fresh microcentrifuge tube and mix with an equal volume of phenol/chloroform/isoamyl alcohol to extract the DNA

-Centrifuge at full speed for 5 minutes

-Transfer the supernatant to a fresh micro centrifuge tube and mix 0.6 mL isopropanol to continue to extract the DNA (A stringy white DNA precipitate will become visible)

-Heat seal and bend a micropipet in a bunsen flame, then use it to hook the DNA and transfer it to a new microcentrifuge tube containing 70% ethanol to wash it

-Centrifuge at full speed for 5 minutes

-Remove the supernatant and dry the DNA pellet in a lyophilizer

-Redissolve the pellet in 100 microliters of TE buffer

References

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