Mini prep and restriction digest conformation

Monday, June 07, 2004

Description
This precedure describes a method for preparation of plasmid DNA and confirmation by restriction endonuclease digestion. See also protocol for cloning of DNA fragments.

Procedure
1 Grow bacterial colonies on agar plates with the appropriate antibiotic until approximately 1mm in size.
2 Using a sterile toothpick or tip transfer a small amount of a single colony to a patch on an agar plate with the appropriate antibiotic. Incubate at 37 degrees.
3 Add the remainder of the colony to 5 ml LB media with the appropriate antibiotic. Grow overnight at 37 degrees.
4 Use approximately 3 ml of culture for the mini prep. Remove media and leave pellet as dry as possible using a pipette and tissue.
5 Resuspend the pellet in 100 ul of ice cold solution one. Leave at room temp for 5 mins.
6 Add 200 ul freshly prepared solution 2. Mix by inversion several times. The solution should go clear. Leave at room temp for 5 mins.
7 Add 150 ul ice-cold solution 3. Mix by inversion, leave on ice for 5 mins.
8 Spin at 4 degrees for 5 ins. This precipitates the proteins and chromosomal DNA leaving only RNA and plasmid DNA in the supernatant. Aliquot the supernatant into a fresh tube.
9 Add equal volumes (approx. 450 ul) of 1:1 or 5:1 phenol:chloro or 25:24:1 of phenol:chloro:isoamylalcohol. Vortex and spin for 5 mins. Aliquot supernatant into a fresh tube.
10 Add 2 volumes of 100% ethanol at room temperature and leave at room temperature for a couple of mins.
11 Spin for 5 mins, pour off the supernatant.
12 Add 1ml of 70 % ethanol, vortex, spin for 5 mins and remove the supernatant.
13 Resuspend the pellet in 20-50 ul TE.
14 Add 1 ul RNAse to digest the RNA leaving only plasmid DNA.

Restriction digest to confirm.

1 Add together: 0.5-1 ul of the appropriate enzyme, 2 ul of the appropriate 10 X buffer, 2 ul of the mini prep DNA and make up to 20 ul with water.
2 Incubate at 37 degrees for 2-3 hours.
3 Run on a 1% agarose gel.
4 Run uncut as well as cut DNA as a control.
5 Check that the appropriate sizes have been obtained to confirm presence of plasmid DNA.

Recipes
Solution one: 50mm glucose, 10mM EDTA, 20mM Tris (pH 8), 4mg/ml lysozyme ( add just prior to use)

Solution two: 2ml 10 M NaOH, 10ml 10 % SDS, 88 ml water.

Solution three : 29.42g KAc, 11.5 ml glacial acetic acid in 100 ml water. (this gives 3M K and 5M acetate)

TE buffer: 10mM tris, 1mM EDTA (pH 8).

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Mini prep and restriction digest conformation