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 mlThe 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 mlThis 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 mlFor 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 ulThe 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 ulTo 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/bagBulk 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):