Gel Extraction of DNA fragment in Agarose

Equipment:
Microcentrifuge
Micropipetters
Water bath 50 C

Starting Material:
DNA fragment in 1% agarose

Reagents and disposable Materials:
clean disposable blade
DNA isolation spin column
1.5 ml microcentrifuge tubes
2 ml microcentrifuge tubes
Micropipette tips
gel dissolving buffer
isopropanol
salted ethanol
sterile deionized water

1) Excise the DNA fragment in 1% agarose with a clean disposable blade (razor blade or scalpel).
Trim to reduce agarose without DNA. This should be ~ 200 mg or less.
2) Add 600 ul of gel dissolving buffer.
3) Incubate @ 50 C for 10 minutes.
If gel is not completely dissolved, mix and incubate for an additional 10 minutes.
4) Add 200 ul of isopropanol.
5) Load DNA isolation spin column into 2 ml tube.
6) Add DNA (in dissolved agarose) solution to column and centrifuge for 1 minute @ 12 K.
7) Add 0.5 ml of gel dissolving buffer and centrifuge for 1 minute @ 12 K to remove all remaining agarose.
8) Add 0.75 ml of salted ethanol and centrifuge for 1 minute @ 12 K to rinse the column.
9) Discard the flow through and centrifuge for 1 minute @ 12 K to remove the remaining ethanol.
10) Load spin column into 1.5 ml microcentrifuge tube.
11) Add 50 ul (or less) of sterile deionized water (or TE) to column, stand for 1 minute at room temperature and centrifuge for 1 minute @ 12 K to elute the DNA.