Taq Cycle Sequencing Reagent Preparation

1. 5X Taq Reaction buffer
	400 mM Tris-HCl, pH 9.0   16 ml  1 M Tris-HCl, pH 9.0
	100 mM (NH4)2SO4, pH 9.0   4 ml  1 M (NH4)2SO4, pH 9.0
	 25 mM MgCl2, pH 7.0       1 ml  1 M MgCl2, pH 7.0
	 5% DMSO                   2 ml  DMSO
				17 ml	ddH2O
				40 ml
The 5X Taq reaction buffer will be added separately with the A, C, G, and T nucleotide mixes for ease in reaction pipetting. One 40 ml preparation of buffer will be sufficient for one batch (about 200 tubes) of A, C, G, and T mix aliquots.

2. Taq Dilution Buffer

	400	mM Tris-HCl, pH 9.0	16 ml	1 M Tris-HCl, pH 9.0
	100	mM (NH4)2SO4, pH 9.0	4 ml	1 M (NH4)2SO4, pH 9.0
	25	mM MgCl2, pH 7.0	1 ml	1 M MgCl2, pH 7.0
					19 ml	ddH2O
					40 ml
This is routinely distributed into 30 ul aliquots in clear, unlabeled 0.5 ml microcentrifuge tubes (about 200 per batch).

3. 50:1 TE

     50 mM Tris-HCl, pH 7.6		0.5 ml 1 M Tris-HCl, pH 7.6
      1 mM Na2EDTA, pH 8.0		0.1 ml 0.1 M Na2EDTA, pH 8.0
					9.6 ml ddH2O
					10 ml

4. Fluorescent Labeled Primers

Prepare a 100 X stock solution (40 uM); an example calculation for a dry tube of an 18mer with an O.D. of 1.00 is shown below (remembering that Joe is the dye-labeled primer for the A reaction, Fam is for C, Tamra is for G, and Rox is for T):

1.00 OD(37 ug/OD)(mol x mer/320 g)(10+12pmol/mol)(g/10+6ug)

(1/18 mer)(ul/40 pmoles)=x ul

In this example x=160 ul, and 160 ul of ddH20 should be added to the dried tube of fluorescent primer for a concentration of 40 uM (40 pmol/ul). From this 100X stock of 40 uM make 1:100 dilutions. To make the amount of primer aliquoted the same as the amount of mixes per batch:

Dilute either fluorescent forward or reverse primers as follows:

	64 ul of 40 uM A or C primer	128 ul of 40 uM G or T primer
	6.3 ml ddH2O  			12.7 ml ddH2O
	6.4 ml				12.8 ml
	
For the A and C primers, distribute the 1X (0.4 uM solution) into 30 ul aliquots, and for the G and T primers, distribute them into 60 ul aliquots. The primers aliquots are stored in clear 0.5 ml microcentrifuge tubes which are labeled with blue, green, red, or yellow markers for A, C, G, or T primers, respectively. (Note: The current primers work optimally at the effective concentration of 0.4 uM, however with each new fluorescent primer preparation, the optimal concentration must be determined). The primers should be stored at 20 or -70degC.

5. 5X Taq Cycle Sequencing Mixes Working dilutions of 20 mM are made for dATP, dCTP, and dTTP based on using one complete tube of 20 mM stock per batch of mixes. 7deaza-dGTP is purchased at a concentration of 10 mM (1080 ul are needed for one batch each of A, C, G, and T mixes, so slightly more than five tubes will be needed-each tube contains 200 ul).

	20 mM dATP		20 mM dCTP		20 mM dTTP
	95 ul of 100 mM dATP	95 ul of 100 mM dCTP	80 ul of 100 mM dTTP
	47.5 ul of 50:1 TE	47.5 ul of 50:1 TE	40 ul of 50:1 TE
	332.5 ul ddH2O		332.5 ul ddH2O		280 ul ddH2O
	475 ul			475 ul			400 ul

The concentration of deoxy and dideoxy nucleotides in the mixes are shown below, followed by the recipe for one 200 tube batch of each of the four mixes.

		A	C	G	T
dATP		62.5 uM	250 uM	250 uM 	250 uM
dCTP		250 uM 	62.5 uM 250 uM 	250 uM
7-dGTP	375 uM 	375 uM	94  uM 	375 uM
dTTP		250 uM 	250 uM	250 uM	62.5 uM
ddATP		1.5 mM	--	--	 --
ddCTP		--	0.75 mM	--	 --
ddGTP		--	--	0.125 mM --
ddTTP		--	--	--	1.25 mM
	
	For one batch (200 tubes) of each nucleotide mix:

		A	C	G	T
20 mM dATP	20 ul	80 ul	160 ul 	160 ul
20 mM dCTP	80 ul 	20 ul 	160 ul 	160 ul
10 mM 7-dGTP	240 ul 	240 ul	120 ul 	480 ul
20 mM dTTP	80 ul	80 ul	160 ul	40 ul
5 mM ddATP	1920 ul	--	--	--
5 mM ddCTP	--	960 ul	--	--
5 mM ddGTP	--	--	320 ul	--
5 mM ddTTP	--	--	--	3200 ul
50:1 TE	640 ul	640 ul	1280 ul	1280 ul
ddH2O		3420 ul	4380 ul	10600 ul7480 ul
		6400 ul	6400 ul	12800 ul12800 ul
To each of these mix solutions, and equal volume of 5X Taq reaction buffer is added (with DMSO), so 6.4 ml is added to A and C, and 12.8 ml is added to G and T. This mix/buffer solution is distributed into 0.5 ml colored microcentrifuge tubes (blue for A, green for C, purple for G, and yellow for T) in 60 or 120 ul aliquots (60 for A and C/120 for G and T). The simplest way to distribute the 60 ul aliquots is 2 x 30 ul using the Eppendorf repeat pipettor set on 3 with the 0.5 ml Combitips, and for the 120 ul aliquots use 1 x 100 ul with the 5 ml Combitip plus 1 x 20 ul with the 0.5 ml Combitip. The mixes should be stored at -20 or -70degC.

Ordering information:

100 mM dATP	27-2050-01   $48   Pharmacia	25 umoles	250 ul
100 mM dCTP	27-2060-01   $48   Pharmacia	25 umoles	250 ul
10 mM c7dGTP	988 537	     $98   Boehringer	2 umoles 	200 ul
100 mM dTTP	27-2080-01   $48   Pharmacia	25 umoles	250 ul
5 mM ddATP	27-2057-00   $25   Pharmacia	0.5 umoles	100 ul
5 mM ddCTP	27-2065-00   $25   Pharmacia	0.5 umoles	100 ul
5 mM ddGTP	27-2075-00   $25   Pharmacia	0.5 umoles	100 ul
5 mM ddTTP	27-2085-00   $25   Pharmacia	0.5 umoles	100 ul
Micro PCR tubes	1044-20-0 $90  Robbins	1000/bag 10 rxn/bag
StripEase caps	1044-10-0 $65  Robbins	 300/bag 25 rxn/bag
Bulk reagents from Pharmacia (cust. no. 6933) (1-800-526-3593) are ordered, with the usual $750 ceiling, and these bulk orders sometimes require a week or two to be filled. Reagents from Boehringer Mannheim (cust. no. 66155-01) (1-800-262-1640) are usually processed overnight. Cycle sequencing tubes from Robbins Scientific (cust. no. 19800-3) (1-800-752-8585):


Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu