Thermal cycler sequecing...

[John Nash]

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.

---
Reagents:

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
		 sequenced.
  -  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
	micropipetting.

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
	following As.
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
cycler.


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!