| Double stranded DNA sequencing (Dideoxy) Template Preparation - For mini prep DNA add 10-19ul (mini prep) DNA to ependorf 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).
- Denature dsDNA with 1ul 4M NaOH and DDW to a total vol of 20ul. Incubate for 5' 37°
- Precipitate with 11.5ul 4M NH4Ac and 84ul ethanol, 5' spin 13K and 70% ethanol rinse.
- 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 - Aliquot 2.5ul (per reaction) of the T C G A termination (dideoxy) mixes into 'V' bottom microtitre tray (Nunc, non-tissue culture treated)
- Make up reaction mix - as a cocktail:
| | 5 clones | 10 clones | | 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 | - Aliquot 5.5ul of the above cocktail to each annelid template half way down tube (ie. above reaction). Pulse spin so that reactions all start at the same time. Vortex. The duration of this prelabelling reaction should be 3-5' (see next step).
- 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.
- Incubate for 5' 37°C. Add 4ul stop solution onto shoulder of well and tap down.
- 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).
- Pre-run gel at "constant power" 55W 15' (see below for gel preparation).
- 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 | | | - | Labelling 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 | | |
