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sect15
Template Preparation
1. For mini prep DNA add 10-19ul (mini prep) DNA to eppendorf with 1ul 10 mg/ml RNASE A and incubate for 10' 37¡C. (For CsCl purified DNA use ~10ug. The RNAse step is not necessary).
2. Denature dsDNA with 1ul 4M NaOH and DDW to a total vol of 20ul. Incubate for 5' 37¡
3. Precipitate with 11.5ul 4M NH4Ac and 84ul ethanol, 5' spin 13K and 70% ethanol rinse.
4. Resuspend in:
2ul sequenase buffer }
3ul primer (10ug/ml) } for each reaction. [Prepare as a cocktail if many reactions]
5ul H20 }
Anneal primer for 30' 37¡C (or longer)
Sequenase Protocol
1. Aliquot 2.5ul (per reaction) of the T C G A termination (dideoxy) mixes into 'V' bottom microtitre tray (Nunc, non-tissue culture treated)
2. Make up reaction mix - as a cocktail:
5 clones 10clones
Labelling Mix (LM) 5ul 10ul
DDW 10ul 20ul
Enzyme dilution buffer 10ul 20ul
0.1M DTT 6ul 12ul
35S dATP 3ul 6ul
T7 DNA pol. 1.5ul 3ul
3. Aliquot 5.5ul of the above cocktail to each anneled template half way down tube (ie. above reaction). Pulse spin so that reactions all start at the same time. Vortex. The duration of this prelabeling reaction should be 3-5' (see next step).
4. Aliquot 3.5ul onto the shoulder of each of the T C G A wells for a given clone, ie just above dideoxy mixes. Cover plate with a lid and tap or centrifuge the reaction down into the dideoxy mixes after 3-5' has elapsed since the pulse spin which first mixed the annealed template with the cocktail.
5. Incubate for 5' 37¡C. Add 4ul stop solution onto shoulder of well and tap down.
6. The sample can be frozen now or proceed with the gel. Immediately before loading incubate for 3' 70¡-90¡C with the lid off (evaporation concentrates the sample). Avoid boiling (100¡ will deform the tray and water may splash into tray).
7. Pre-run gel at "constant power" 55W 15' (see below for gel preparation).
8. Load 1.7ul to each track after flushing wells. Run till bromophenol blue is near the bottom, ~ 3hrs. Dry for ~ 1hr. A.R. o/n R.T.
Sequenase buffer: 5 X
200mM Tris HCl pH 7.5
100mM MgCl2
250mM NaCl
Labeling mix
7.5uM dGTP
7.5uM dCTP
7.5uM dTTP
ddG termination mix
80uM dGTP, 80uM dCTP, 80uM dTTP, 80uM dATP
8uM ddGTP, 50mM NaCl
ddC termination mix
80uM dGTP, 80uM dCTP, 80uM dTTP, 80uM dATP
8uM ddCTP, 50mM NaCl
ddT termination mix
80uM dGTP, 80uM dCTP, 80uM dTTP, 80uM dATP
8uM ddTTP, 50mM NaCl
ddA termination mix
80uM dGTP, 80uM dCTP, 80uM dTTP, 80uM dATP
8uM ddATP, 50mM NaCl
Enzyme dilution buffer
10mM Tris HCl pH 7.5
5mM DTT
0.5mg/ml BSA
Stop Solution
95% formamide
20mM EDTA
0.05% bromophenol blue
0.05% Xylene Cyanol
ALF sequencing with fluorescein labelled nucleotides.
1. Denature, precipitate and anneal primers to template DNA by standard method.
2. Add 2µl of labelling mix to the annealing reaction and begin labelling reaction by adding 2µl of diluted T7 polymerase. Incubate 10 minutes at 37ûC.
3. After the labelling reaction is complete, add 1µl extension buffer and 3µl DMSO. Proceed to the termination reactions as per the standard protocol.
Fluoro nucleotides for non-labled primers.
fluoro-dATP labelling mix: 10µM fluorescein-15-dATP
1µM each dCTP, dGTP, dTTP.
fluoro-dUTP labelling mix: 10µM fluorescein-12-dUTP
1µM each dCTP, dATP, dGTP