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% eth

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