DNA Isolation

1. Inoculate 3 mL-portions of LB containing 20 ^g/mL tetracycline with positive clones identified in Subheading 3.4. Shake the tubes vertically for 16-24 h at 37°C.

2. Microfuge each culture briefly to pellet cells.

3. Pipet supernatant into a vessel containing 450 ^L of PEG/NaCl. Mix by 100X invertions and incubate on ice for at least 4 h.

4. Microfuge for 15 min, remove supernatant, recentrifuge briefly, and remove supernatant.

5. Dissolve pellet in 500 ^L of TBS by vortexing. These phage are already pure enough for preparing sequencing templates.

6. Put 500 ^L of PEG-purified phage suspension into Eppendorf tube. Add equal volume of phenol/chloroform. Vortex vigorously. Microfuge to separate phases. Collect the upper aqueous phase (approx 400 ^L) trying to avoid any traces of interphase and lower phase.

7. Transfer aliquot to a second Eppendorf tube with 40 ^L of 3 M sodium acetate and 1 mL of ethanol. Allow DNA to precipitate for at least 1 h on ice.

8. Microfuge for 15 min. Remove supernatant, recentrifuge briefly, and remove supernatant again.

9. Wash pellet (usually invisible) with 1 mL of 70% ethanol. Remove supernatant, recentrifuge briefly, and remove supernatant.

10. Dissolve pellet in 8 ^L of water.

11. Run 1 ^L of sample in 1% agarose gel to control purity of DNA. To obtain good sequence results, a sharp band of around 9000 bp should be visible.

Boil 1 g of molecular biology grade agarose in 100 mL of 1X TBE buffer in a glass conical flask until a clear transparent solution is achieved. Cool to 60°C, add 5 ^L of ethidium bromide (10 mg/ mL), and mix thoroughly. Pour into gel apparatus with sample comb and leave to set for 20 min to allow the gel to set. Add just enough electrophoresis buffer (1X TBE) to cover the gel. Mix phage DNA samples with an equal volume of 2X agarose gel sample buffer and load 10 ^L of each into the slots of the submerged gel. Run gel at 200 volts for 11-15 min and examine with a UV transilluminator. The rest of the DNA can be stored at -20°C for a few weeks.

Was this article helpful?

0 0

Post a comment