summary Double Stranded DNA Sequencing

[penas at ucbeh.san.uc.edu]

Hi! I want to summarize what I have been doing for my double stranded
sequencing reactions.  Sorry it took me so long but I was out of town
for a conference during the last two weeks.  As I mentioned in a
previous mini-summary the most critical aspect seems to be the
cleanliness of the template DNA to be sequenced.  The following is the
miniprep protocol that I am using which is from INVITROGEN:

		MINI Plasmid Preparation Protocol
(Reference:  Zhou et. al., Biotechniques Vol 8., No. 2:172 (1990))

1.	Grow 2 mls of bacterial culture at 37 C overnight.
2.	Spin 1.5 mls of the culture in a microcentrifuge tube for 10
	sec, decant supernatant leaving 50-100 ul of medium in the tube.  Vortex
	to resuspend cells completely.
3.	Add 300 ul of TENS (10mM Tris-HCl, pH 7.5, 1mM EDTA, 0.1N NaOH,
	0.5% SDS), vortex for 2-5 sec.
4.	Add 150 ul of 3.0M Na (or K) Acetate, pH 5.2, vortex for 2-5 sec.
5.	Spin for 2 min to pellet cell debris and chromosomal DNA. 
	Transfer supernatant (just 300 ul) to a fresh tube, add 900 ul 100%
	ETOH and vortex.  Freeze at -70 C for 30 min.
6.	Spin 5 min to pellet plasmid and RNA.  The pellet should have a
	white appearance.  Discard supernatant and wash pellet twice with 1 ml
	of 70% ETOH.  Remove any residual ETOH.
7.	Resuspend pellet in 20-50 ul of sterile H2O.

Note that this protocol does not require phenol:chloroform extractions
or RNAse treatment.  

Following this procedure I clean my plasmid DNA with prep-A-Gene
(BIORAD).  Prep-A-Gene gets rid of RNA and any contaminant on your DNA
sample.

The next step is to denature the plasmid DNA.  I have been using a
modification of a protocol sent by William J. Buikema of the Univ. of
Chicago.
			Alkaline Denaturation:
1.	ETOH precipitate the DNA obtained from PREP_A_GENE (I use large
	amounts of elution buffer to increase my yield.
2.	Dissolve pellet in 20 ul of Sterile H2O and add 2 ul of freshly
	prepared (I don't know how critical is it to use freshly prepared
	solution since I did all my sequencing reactions (20) at once, and I
	haven't repeated any yet) 2 N NaOH, 2 mM EDTA.  Mix well and incubate
	for 30 min at 37 C.
3.	Add 3XETOH (100%) mix and incubate for 20 min at -70 C.


The next step is to do the sequencing reactions.  I followed the
protocol of Sequenase version 2.0 (using their Manganese buffer).

My results have always (consistently) been optimum.  I am able to read
200-250 bases.
-- 
Sandra Pena de Ortiz     : penas at ucbeh.bitnet
University of Cincinnati
College of Medicine
Toxicology Program