Cycle sequencing anyone?

Alexander Kraev kraev at bc.biol.ethz.ch
Mon Mar 24 06:16:59 EST 1997


In article <3334A8B1.15BC at utoronto.ca>, Ed Marsden
<ed.marsden at utoronto.ca> wrote:

> Netters,
> 
> For the sake of simplicity, I've attempted to cycle sequence both the 
> commercial plasmids and my own ds isolate as opposed to using the 
> standard protocol (we're using the Boehringer Taq polymerase kit and DIG 
> detection).

I do not understand what do you mean by "simplicity" here.  Cycle sequencing
with hapten detection is not a simpler procedure as opposed to, say, non-cycled
radioactive label sequencing.  It is non-radioactive, but has more steps
than can
go wrong than the radioactive procedure has. 

> 
>         Here's the scoop: After X-ray development, we cannot see ANY 
> bands - only background. The first x-ray was pretty dark so we cut the 
> exposure time in half which gave us less blackened background but still 
> no ladder (except mabye the wells?)
>         My professor and I agree that the problem may be due to the 
> sequencing reactions (I've followed the Boehringer protocol 
> religiously for all steps). I'm wondering if it would be advisable to 
> boost the concentrations of template, buffer, primer et al. and add more 
> dH2O to reduce the viscosity of the mixes? Increase the number of 
> cycling steps? 
>    I don't believe that the detection protocol is awry as we do get 
> residual background due to the chemiluminescence (blotches on the film 
> due to a poor blocking job). The nylon transfer seems to be working well 
> also...

However, first you should troubleshoot the detection, and then the sequencing
reaction, in that order.

1. Spot your primer on your membrane and run the detection procedure as you
do it (same reagents, times, trays/ bags).  This membrane should reveal very
dark   primer spots after 1 minute exposure, but the rest of it should be BLANK.
If you don't get such a result,  you should check your  membrane, reagents and
the trays/bads you are using.  If you result is different, the detection
does not work.
Obtain a Boeringer booklet on DIG, it has extensive description of  what can
go wrong with the detection.

2. After you have got a blank membrane with primer spots, expose it for 1 hour.
You should get  VERY LARGE dark spots on uniform light grey background.  Now you
are ready to troubleshoot your sequencing reaction. 

3. Make up  control DNA sequencing reaction as indicated in the kit  (i.e.
1 ug template
and 2-5 pmol primer) and run 30-50% of it on a  seq. gel ( you need a 20
cm gel to troubleshoot,
 do not go for a real one).  Transfer and detect it as above. After 20-60
min of exposure
 (depending on the film), you should see your control ladder on light grey
background.  If you do not
see any bands on light grey background,   check your cycler.  The
temperature display may be
way off real .  Your should be able to get a  ladder from control DNA 
when cycling at the following
conditions:  20X(30 sec 94 C,  1 min 50 C, 2 min 70 C).  Many newer
cyclers allow for
much shorter times, but this one should work on any machine. 

Good luck,

-- 
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch



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