DNA Preparation - Bacterial DNA in Agarose Plugs


Jeff Lawrence

1. Grow 5 mL culture overnight. Pellet cells at 8K for 10 min and resuspend in 600 µL PIV:
	10 mM	Tris, pH 8.0	(1 mL	1 M Tris, pH 8.0)
	1 M	NaCl		(20 mL 5 M NaCl)
	250 mM	EDTA		(50 mL 500 mM EDTA, pH 8.0)
				(29 mL	ddH2O)
2. Mix with 600 µL 1.3% plug-quality LMT Agarose at 42°. Mix quickly and dispense 110 µL into plug moulds (Pharmacia). Place moulds at 4° for 20 min to solidify.

3. Remove plugs from moulds by ejecting with a pipet bulb into a small quantity of liquid. Place in 20 volumes of:
	10 mM	Tris, pH 8.0	(1 ml	1 M Tris, pH 8.0)
	1 M	NaCl	20 ml	(5 M NaCl)
	250 mM	EDTA	50 ml	(500 mM EDTA, pH 8.0)
	0.5 %	Sarkosyl	(5 ml	10% Sarkosyl)
	0.2%	Deoxycholate	(4 ml	5% Deoxycholate)
	20 mg/ml DNase-free RNase	(2 mg	RNase)
	1 mg/ml	Lysozyme	(100 mg	Lysozyme)
				(20 ml	ddH2O)
4. Incubate overnight at 37° with slow shaking. Hard shaking will damage plugs. Decant rinse with Pasteur pipet (use vacuum with caution) and add 20 volumes of:
	250 mM 	EDTA	50 mL	(500 mM EDTA, pH 8.0)
	1 % 	Sarkosyl	(10 mL	10% Sarkosyl)
	1 mg/ml	Proteinase K	(100 mg	Proteinase K)
				(40 mL	ddH2O)
5. Incubate for 24-48 hours at 42-50°. Decant rinse and place in 10 volumes of:
	10 mM	Tris, pH 8.0	(1 mL	1 M Tris, pH 8.0)
	1 mM	EDTA	200 mL	(500 mM EDTA, pH 8.0)
	1 mM	PMSF 	1 mL	(100 mM PMSF)
				(98 mL	ddH2O)
6. Incubate at room temperature for 1-2 hours. Change rinse at least three times with fresh solution.

7. Decant rinse and begin rinsing with large volumes of TE10:
	10 mM	Tris, pH 8.0	10 mL	(1 M Tris, pH 8.0)
	10 mM	EDTA, pH 8.0	20 mL	(500 mM EDTA)
					(970 mL	ddH2O)
8. Change rinse every 2-6 hours. Rinse at least ten times. Patience is a virtue.


Last Update: Thursday, 19-Jun-2014 11:37:52 PDT
This page has been viewed 282 times.
Eric Kofoid eckofoid at ucdavis.edu