Thermal cycler sequecing...
num208jn at MBDS.NRC.CA
Tue Apr 7 08:48:16 EST 1992
In article <1992Apr1.170103.257 at nrcnet0.nrc.ca>, I wrote:
>In article <9203312113.AA25586 at umailsrv0.UMD.EDU>, BILL wrote:
>>Does anyone out there get good results from thermal cycler sequencing ?
>>We have been trying to sequence gel isolated PCR products using
>>a gel purified end labelled primer (one of the PCR primers) and
>>can't get clean sequence. We've tried 3 step and two step cycling
>>with various time regimes and temperatures and still no luck.
>>Has anyone had better luck using this technique ???
>I have never used Taq polymerase based cycle sequencing, but I have
>had excellent results sequencing a 1 kb PCR product with New England
>Biolab's CircumVent sequencing which utilises exo-minus Vent
>polymerase. I use their standard reaction protocol but cycle at
>95/1min; 55/1min; 72/1min; twenty times after a hot start.
>My product was excised from an agarose gel, cleaned up with Glass
>Milk, and eluted into water.
>If you want further details... email me.
>P.S. Usual disclaimer...
Ok folks... I got lotsa email requests to post the method for
CircumVent sequencing. My original email responses to queries could be
summarized by the following...
"I'm not doing anything special - I deserve NO credit for this,
I using New England Biolab's CircumVent kit almost exactly as they
direct EXCEPT that I'm doing 20 cycles at 95 deg/ 1 min; 55 deg/
1min; 72 deg/ 1 min, instead of the 20 sec they recommend.
I phoned them and they faxed me the protocol I use - maybe they'll
send it to you."
Since I got lots of email for more details incl. the protocol, I'll
post them here (in lieu of replies by email).
The cycle sequencing kit is approx Cdn$150 for 50 reactions, or the Vent
pol exo-minus enzyme is Cdn$84 for 100 units.
Protocol (disclaimer... it's probably copyright NEB, it's from the sheet
they faxed me): THIS IS FOR VENT POL exo-minus. WHO KNOWS WHAT WILL
HAPPEN WITH TAQ POL!!!
One can use a 5' end labelled primer or labelled dATP incorporation. I
have only done the latter. Apparently with labelled primers, you only
need minute amounts of template, e.g from colonies, plaques, etc.
I use 35S dATP (c1500 Ci/mmol) as label.
a. 10X CV sequencing buffer: b. 30X TritonX-100:
100 mM KCl 3% Triton-X-100
100 mM (NH4)2 SO4
200 mM Tris-HCl, pH 8.8
50 mM MgSO4
c. CV Deoxy/dideoxy mixes (uM concentrations):
A MIX: C MIX: G MIX: T MIX:
ddATP 900 - - -
ddCTP - 600 - -
ddGTP - - 500 -
ddTTP - - - 800
dATP 30 30 30 30
dCTP 100 30 100 100
dGTP 100 100 30 100
dTTP 100 100 100 30
Made up in 1 X CV sequencing buffer...
d. Usual formamide stop dye with BPB, XC. etc...
Cycle sequencing with dATP incorporation:
1. Add 3 ul A mix to a tube labelled A, 3 ul C mix to a tube labelled C,
etc... (Use the size of tube which fits in your cycler!).
2. Mix the following in a microfuge tube:
- 0.04 pmol ssDNA or 0.1 pmol dsDNA
- 0.6 pmol primer if ssDNA or 1.2 pmol primer if dsDNA is being
- 1.5 ul CV 10x sequencing buffer
- 1.0 ul 30X triton-x-100 buffer
- ddH2O to make volume to 12 ul.
3. To the template/primer mix, add 2 ul alpha-35S dATP (500-1200
Ci/mmol). [I got ok results with 1 ul, but had to expose the
autorad for 24 hr -JN]
4. Add 2 units of Vent pol exo-minus. Mix the reaction mix by gentle
5. Distribute 3.2 ul of this to the tubes marked A, C, G and T, and
overlay with a drop of oil.
6. Cycle at 95 deg; 55 deg; 72 deg. With Techne PHC-2 cyclers, they
recommend 20 cycles of 20 sec each temp. With Perkin Elmer Cetus 480s,
they recommend 25 cycles of 20 sec each temp. With my COY model 60
TempCycler, I have to use 20 cycles of 1 min each temp for dsDNA to
give decent bands.
7. Underlay 4 ul of stop solution... store, heat, run etc.
NOTE: For you Sequenase users... it's like Klenow time all over again
because the bands have shade characteristics ;-(
a. the first C in a run of Cs is darker than the following Cs.
b. the second A in a run of As is darker than the preceding and/or
c. G following A tends to be darker than following Gs.
d. T following A tends to be darker than following As.
Hope this is useful... Sorry for any typos...
DISCLAIMER: I have nothing to do with New England Biolabs. They have
been extremely helpful to me to get this protocol working with my
John Nash (Internet:) Nash at biologysx.lan.nrc.ca.
Email to my other addresses is forwarded automatically,
Disclaimer: All opinions are mine, not NRC's!
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