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MOLECULAR BIOLOGY: WORKING WITH DNA

SEQUENCING

Extended Sequencing with Klenow Fragment

Extended Sequencing with Klenow Fragment Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Andrew Murray at Harvard University
Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Annealing the primer 1. Add the following to a small microcentrifuge tube 5 μl M13 DNA (1 μg with a standard 1.5 ml pellet in 25 μl; equals 0.4 pmol) 2 μl sequencing Buffer 3 μl 17-base primer (2 μg/ml; equals 0.5 pmol) Add ddH2O to 10 μl final volume 2. Mix contents and incubate 5 min at 65°C 3. Cool slowly to room temperature (10 min) 4. Prepare microtiter plate with wells labeled T, C, G, and A containing 2 μl each of the corresponding termination mix. B. Labeling-extension reaction 1. Centrifuge the template primer tube and then add: 2 μl freshly-diluted 1X Extension Mix 1 μl sequencing label (10 mCi/ml α-[35S]dATP at 600 Ci/mmol; CAUTION! see Hint #1) 1 μl 100 mM DTT 2 Units of Klenow DNA polymerase Add ddH2O to 16 μl total 2. Mix, centrifuge and incubate 5 min at room temperature (20°C). C. Termination reaction 1. Transfer 3 μl of the extension reaction to each of the termination mix wells. 2. Mix the contents well. 3. Incubate the plate for 10 min at 37°C. 4. Add 4 μl of Formamide Dyes to each well. 5. Heat to 80°C for 5 min. 6. Load 2 μl of each reaction on the sequencing gel if a 6-tooth comb is used, or load 1 μl for a 12-tooth comb.
Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
50 mM Stocks of Nucleotides    Information given as: Base, wavelength, e(M-1 cm-1), absorbance of 1:800 diluted 50 mM solution T, 260, 7.4 X 103, 0.463 U, 262, 1.0 X 104, 0.625 C, 271, 9.1 X 103, 0.569 G, 253, 1.37 X 104, 0.856 A, 259, 1.54 X 104, 0.963 Sequencing Buffer (5X)    50 mM MgCl2 250 mM NaCl 200 mM Tris, pH 7.5 100 mM DTT TBE Buffer (10X)    890 mM Boric Acid 890 mM Tris 20 mM EDTA pH to 8.0 6% Acrylamide/Urea Gel Mixes    1X TBE, 75 ml, 50 ml, 230 g, 200 ml, 500 ml TBE concentration, 38%:2% Acrylamide:Bisacrylamide (CAUTION! see Hint #1), 10X TBE, urea, water, total volume Information given as: 0.5X TBE, 75 ml, 25 ml, 230 g, 225 ml, 500 ml 5X TBE, 30 ml, 100 ml, 92 g, 0 ml, 200 ml for TBE gradients Formamide Dyes    0.05% w/v Xylene Cyanol FF 20 mM EDTA 95% v/v Deionized Formamide (CAUTION! see Hint #1) 0.05% w/v Bromophenol Blue Termination mix    Information given as: T, 5, 50, 50, 50, 100, 1745 A, 50, 50, 50, 5, 60, 1785 5X extension mix, 1.5, 1.5, 1.5, 0, 0, 1995 Mix, dTTP, dCTP, dGTP, dATP, ddNTP, water C, 50, 5, 50, 50, 20, 1825 (volumes (μl) of 10 mM stocks for 2 ml) G, 50, 50, 5, 50, 30, 1815 Termination mixes    Information given as: T, 25, 250, 250, 250, 500 A, 250, 250, 250, 25, 300 C, 250, 25, 250, 250, 100 G, 250, 250, 25, 250, 150 all numbers represent μM concentration Mix, dTTP, dCTP, dGTP, dATP, ddNTP Extension Mix (5X)    7.5 μM dTTP 7.5 μM dGTP for α-[35S]dATP as label 7.5 μM dCTP
BioReagents and Chemicals Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
α-[35S]-dATP Tris Formamide, Deionized DTT Boric Acid Acrylamide Xylene Cyanol FF Bromophenol Blue Sodium Chloride EDTA dCTP dTTP dGTP dATP Klenow Fragment Magnesium Chloride Oligonucleotide Bisacrylamide Tris Base Urea
Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

   

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