M13SEQ

M13 SEQUENCING PROTOCOL

COMPLEMENTATION (or "C")-TEST
This assay will allow you to determine if inserts in different M13 ssDNA molecules are in opposite directions. If they are, the inserts will anneal to each other and migrate slower through a gel as a figure 8 shaped molecule, relative to the molecules that have not annealed.

Mix 20 µl of each pair of bacteriophage-containing supernatant with 4 µl of Endo R dye mix. Prepare positive and negative controls in the same manner.
Heat to 65°C for 1 hour and load on a 1% agarose gel.
Run in 1X TBE buffer at 100 volts for 5 hours. Stain the gel with ethidium bromide.

EXTRACTION OF DNA FROM M13 BACTERIOPHAGE (Adjust volumes proportionately as needed).

Centrifuge the supernatant from an overnight (or 6 hour) 1.5 ml culture for 10 minutes at room temperature in a microcentrifuge. (If you are using a large centr ifuge, first centrifuge the sample at 2500 rpm to remove any bacteria.)
Precipitate 0.75 ml of the supernatant with 150 µl of 20% PEG, 2.5 M NaCl, for 15 minutes at room temperature.
Centrifuge at high speed in a microcentrifuge for 5 minutes.
Discard the supernatant and resuspend the pellet in 75 µl of TE.
Phenol extract the supernatant with 37.5 µl of STE-saturated phenol. Vortex the sample for about 2 seconds, wait for 5 minutes, vortex for another 2 seconds, and separate the layers with a 5 minute spin in a microcentrifuge.
Collect, then extract the upper aqueous phase with ether 3-4 times to remove phenol.
Ethanol precipitate the DNA at -70°C for 20 minutes.
Spin for 20 minutes in a microcentrifuge at 4°C. Wash the pellet once with 0.5 ml of cold 80% ethanol. Spin for 5 minutes and dry the pellet under vacuum.
Resuspend in 37.5 µl TE and check for DNA on an ethidium bromide pla te.

PRIMER ANNEALING REACTIONS: 5.5 µl water; 1 µl polymerase reaction buffer concentrate; 1 µl primer (stock concentration is 2.5 ng/ul); 5 µl DNA from above
Heat to 95°C for 5 minutes, then cool slowly to room temperature.

M13 SEQUENCING REAGENTS
Frozen stocks (stored at -30°C): dNTP stocks: 20 mM in water; ddNTP stocks: 10 mM in water; M13 primer: 25 ng/µl in polymerase buffer concentrate

Working Dilutions: M13 Primer: 2.5 ng/µl (1/10 dilution of stock in water - good for 6 mo?); dNTP: 0.5 mM (1/40 dilution of stocks in water; add 2.05 µl stock to 79.95 µl water). These may be stored at -30°C for 2-1/2 to 3 weeks maximum.; ddA & ddT: 1 mM (1/10 dilution of stocks in water); ddG & ddC: 0. 5 mM (1/20 dilution of stocks in water); Store the ddNTP working solutions as dNTPs

Preparation of Sequencing Solutions [for use with a-³²P-dGTP]

Reaction	0.5 mM dTTP	0.5 mM dCTP	0.5 mM dATP	0.5 mM dITP	Pol. Rxn Conc.
A	20 µl	20 µl	1 µl	---	20 µl
C	20 µl	1 µl	20 µl< /TD>	---	20 µl
G	20 µl	20 µl	20 µl	---	20 µl
T	1 µl	20 µl	20 µl	---	20 µl
I	20 µl	20 µl	20 µl	3.2 µl	20 µl

Sequence Reaction Tubes [Approximate proportions - adjust after running gel]: A reaction solution: 1.6 µl + 0.4 µl ddA; G reaction solution: 1.6 µl + 0.4 µl ddG; C reaction solution: 1.7 µl + 0.35 µl ddC; T reaction solution: 1.25 µl + 0.85 µl ddT; I reaction solution: 1.5 µl + 0.55 µl ddG

Sequencing Reaction: Add mixtures of dNTP and ddNTP to the sequence reaction tubes.

RECIPES

Formamide Dye Mix
For 1 ml: 950 µl deionized formamide

30 µl water

20 µl 0.5 M EDTA

1 mg bromophenol blue

1 mg xylene cyanol FF

Mix and store at 4°C.

Endo R Dye Mix: 1 µl 2% SDS; 3 µl of [0.2% BPB, 50% glycerol, 0.2 M EDTA, pH 8.0-8.3 in water].

20% Polyethylene Glycol, 2.5 M NaCl: 25 ml 5 M NaCl; 10 g PEG (mw 8000)

Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF

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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. M13 Sequencing Protocol. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/m13seq.html). 1997. Oklahoma City. Rev ision Date: October 2, 1997."