What's wrong with my DNA sequencing template?

Michel Leroux mleroux at unixg.ubc.ca
Sat Mar 18 15:43:25 EST 1995

In article <binnie_betten-2302950852540001 at dipietro.dept-med.pitt.edu>,
binnie_betten at paccm.pitt.edu (Binnie Betten) wrote:

> This is my first attempt at sequencing, and although I get beautiful
> sequencing info from the control DNA that comes with my sequencing kit, I
> get absolutely no bands of any kind from my single stranded sample DNA.
> Here's what I'm doing:
> 1)  I do an M13 Helper Phage Rescue with grown-up (previously frozen)
> cloned stocks.
> 2)  Followed by a PEG 8000 small scal prep on the infected culture
> 3)  Run ssDNA samples on LMP agrose containing Ethidium Bromide, and use
> "medium-range" UV to detect and excise DNA bands.
> 4)  Purify ssDNA using "Gelase fast protocol."
> 5)  Ethanol precipitate as recommended in Gelase protocol.
> 6)  Quantitate DNA by UV OD260.
> 7)  Use 1 microgram of purified ssDNA as recommended in Amersham's Version
> 2.0 Sequenase Kit (along side of kit DNA controls which work fine).
> So does anyone have any ideas about what could be going wrong.  I thought
> maybe using the medium range UV to excise the bands could be the problem;
> but would a few minutes exposure at this wavelength really make a
> difference?

Why don't you try double-stranded DNA sequencing? It's much quicker and
gives equivalent results.
Here's a brief summary of my method:
-grow 3ml culture, pellet, resuspend in Glucose-EDTA-Tris buffer (200ul),
add 400ul NaOH/SDS, mix, then 300ul 7.5M NH4Oac, on ice for 10 minutes,
centrifuge 5min, take 700ul sup and add 420ul isopropanol, vortex, leave
10min, then centrifuge, wash once with 70% EtOH, air dry, resuspend pellet
in TE-RNAse buffer (50-65ul), and use 15ul for sequencing.

-for sequencing, add 2ul 2M NaOH, 1ul primer (1pmol/ul), leave room temp
for 5min, add 8ul 5M NH4Oac, 100ul 99% EtOH, vortex, put in -70C 15 min,
spin 10min, wash w/ 70% EtOH, air dry 10min, and resuspend in 10ul 1X
sequenase buffer; leave at 37C for 15min, then add 1ul 100mM DTT, 2ul
labelling mix, 0.5ul 35-S dATP, and proceed as you normally would to

Hope this helps,

Michel Leroux
mleroux at unixg.ubc.ca

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