summary Double Stranded DNA Sequencing
penas at ucbeh.san.uc.edu
penas at ucbeh.san.uc.edu
Mon Nov 18 12:56:16 EST 1991
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
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
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
Sandra Pena de Ortiz : penas at ucbeh.bitnet
University of Cincinnati
College of Medicine
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