DNA priming reaction

x ul DNA
2 ul Reaction buffer, minus DTT
1 ul primer (20 ng)
10 ul total
Heat 90-100C 2 min.
Cool room temp. 30 min.

Enzyme mix
4 ul radioactive nucleotide (12 l)
2 ul reaction buffer ( 6 l)
4 ul water (12 l)
4 units enzyme (1tube)

Mix DNA and Enzyme mix

Add 4 ul to 1 ul nucleotides (see nucleotide mixes).
Incubate 50-60C 15 min.
Add 1 ul chase (0.5mM nucleotides)
Incubate 15 min.
Stop and Load
Reaction buffer (5X) per ml
0.3 M Tris (pH 8.3) 300 ul 1M
no NaCl
37.5 mM MgCl2 37.5 ul 1M
5 mM DTT 5.0 ul 1M
water 660 l

SEQUENASE REACTIONS

Mix:
7 ul total of DNA plus water [1-2 g]
2 ul sequenase buffer
1 ul primer
Heat 2 min. at 90-100C
Add 1 ul of 1:4 dilution of s.s. binding protein.
Leave 30 min. at room temperature.

While waiting:
Unfreeze label (35S or 32P dATP)
Unfreeze Sequenase items
Labelling mix (one for dGTP; one for dITP if necessary)
0.1 M DTT
Stop Mix

Termination mixes (one set dGTP; one set dITP if necessary)
Aliquot into tubes marked G,A,T,C:
2.5 ul of appropriate termination mixes.

Mix for dGTP reactions:
11 ul DNA mix
1 ul 0.1 M DTT
2 ul labelling mix (1:5 dilution dGTP mix for reading ~500 bp)
.5 ul dATP -32P or -35S
2 ul 1:8 dilution of Sequenase (dilute with TE)
Wait 5 min. at room temp.

Pre-warm termination mixes to 37C

Add 3.5 ul of labeling mix to each termination mix

For dITP wait 2-3 min. to add 4 ul stop solution.

For dGTP around 5 min. is OK.

This means dITP reactions are best done one at a time.

dGTP reactions can be done three at a time.

Add 1l of a 1:10 dilution of 20 mg/ml Proteinase K.

Heat at 65C for 20 min.

Before loading onto gel heat around 90C and load immediately.

To wash short [notched] plate:
Wash in 2.5% NH4OH, 50% isopropanol, 1/2 cap detergent per 500 ml.
Then 2X with H2O, and 2X with 95% ethanol.
Check for good siliconization; if not good, then siliconize with 2% dichlorodimethylsilene in toluene, and repeat wash with water and ethanol.

To wash long [unnotched] plate:
Wash in 10 N NaOH. Then 2X with H2O, and 2X with 95% ethanol.

Acrylamide solution:

Make 100 ml of 6% (w/v) acrylamide sequencing gel solution with following ingredients:

48 g urea, 30 ml H2O [to mix], 10 ml 10X TBE, 15 ml acrylamide (38%)/bis-acrylamide (2%), 400 ul 40% (w/v) ammonium persulfate, when completely dissolved bring to final volume with water.

Filter solution through 0.2 m filter unit. Cool solution on ice for about 15 min [helps to slow polymerization reaction].

When ready to pour gel, add 60 ul TEMED immediately before casting gel (within 30 sec).

Let gel polymerize for at least 2-3 hours.

Pre-electrophoresis:

Pre-run gel in TBE buffer for 30 min or more at constant power of 45-50 W until temperature reaches 50C.

Electrophoresis:

Run gel at 45-50 W at 50C for about 2.5 hr.

Post-electrophoresis:

Open plates. Gel should stick to long plate. Fix 35S run in 10% (v/v) acetic acid, 5% (v/v) methanol. Transfer to Whatman paper and dry gel for 30 min at 80C. Expose to X-ray film.