Prepare G A T C tubes; one set for each clone to be sequenced. Add 3uL of each
V-1 mix to respectively labeled tubes on ice.
Extension reaction:
Add to annealing reaction:
2.0uL extension mix v (1Xext)
2.0uL 35S-dATP
1.0uL vent (exo-) DNA polymerase (2000u/mL)
Mix.
Incubate 42°C for 5 minutes.
Add 3.2uL of extension reaction to each GATC tube, mix. Incubate at 72°C
for 10 minutes.
Spin and quickly add 4 uL of STOP/LOADING solution.
Samples may be frozen at -20°C overnight before running on the
sequencing gel. Heat samples 2 minutes at 72°C just prior to loading. (In other
words, the samples as well as the sequencing gel should be hot in order to get good
seperation.)
Load 2.5 uL of each GATC sample in that order. If you avoid the outermost lanes, you
can load 13 loads onto one sequencing gel.
NEB SEQUENCING (VENT POLYMERASE)--ALTERNATE METHOD
PREPARING SAMPLES TO BE SEQUENCED
Vectors are double stranded and firstly the DNA must be denatured.
For each tube:
4 M NaOH
1.0 uL
4 mM EDTA
1.0 uL
dH2O
15.5 uL
DNA
2.5 uL
Total = 20 uL
Incubate at room T for 5 minutes.
Ethanol precipitate solution to pellet DNA--gets rid of NaOH in solution.
4uL of 5 M NH4OAc
2X the volume of EtOH(100%)=48uL EtOH (b/c 20uL solution +4 uL salt
(NH4OAc) +24uL)
spin for 15 minutes
Keep the pellet (hard to see), discard supernatant.
Each tube containing pellet was filled with 6.5uL of dH2O to resuspend pellet.
Each tube is heated at 95°C for 5' (to denature DNA since NaOH has been
removed.
After 5' the tubes are placed in an ice bath to "quick-cool" them. (This ensures
denatured DNA will not reanneal)
Anneal DNA to primer.
DNA
6.5uL
10X vent buffer
1.5uL
primer(T7 5pmol/uL)
1.0uL
Total
9.0uL
Incubate at 42°C for 20 minutes.
Extension reaction--add the following to the above;
Ext. Mix
2.0uL
35S dATP
2.0uL
Vent polymerase
1.0uL
DNA + primer solution
9.0ul
Total
14uL
Incubate at 42°C for 5 minutes.
GATC tubes were prepared for each clone
Add 3uL of each dNTP solution into its respective GATC tube.
Add 2.8uL of extension reaction (14uL) for each clone to its respective GATCset of
tubes.
The tubes were then mixed by flicking with finger or vortex, and were then
incubated at 72° C for 10'. (The reaction proceeds very quicly ~
30bp/sec)
The solution were spun and 4uL of STOP/LOADING solution was added to each
solution.
The tubes can then be frozen.
. The samples were heated before loading at 72°C for 2 minutes (or 7 minutes if
previously frozen) , so that they are the same temperature as the gel.
The combs were inserted into the warming gels.
2.0uL of each clones GATC tubes (while still in the heat block) were quickly added
to the wells.
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