Cycle Sequencing with v 3.1 Big Dyes
Cycle Sequencing:
What ABI recommends:
Terminator Ready Reaction mix (Big Dyes)
BigDye Seq Buffer (Dilution Buffer)
Primer
Template
Water
4 l
2 l
x l (3.2 pmol)
y l
z l
Total
20 l
Our recipe:
Terminator Ready Reaction mix (Big Dyes)
BigDye Seq Buffer (Dilution Buffer)
Primer
Template
Water
1-2 l
4l-amt of Big Dyes
x l
y l
z l
Total
10 l
NOTE: Add dilution buffer so total volume of dilution buffer and Big Dyes is 4 l
.5 l of 10mM primer is usually more than enough
Initial denaturation of 96o C for 1 min
96o C for 10 sec
50 o C for 5 sec
60 o C for 4 min
25 cycles total
This is programmed in the main folder on all the thermal cyclers. The program is called
“3_1Auto.”
Purifying products:
There are two suggested alcohol precipitation methods. The first uses EDTA while the other
uses EDTA and sodium acetate. The one that uses EDTA minimizes unincorporated dyes but
may cause loss of smallest fragments. The EDTA/NaOAc method will improve recovery of
smallest peaks but will also increase the amount of residual unincorporated dyes.
METHOD 1: EtOH/EDTA Precipitation in 96-well trays:
1) Make a master mix of EtOH/EDTA solution. If your final sequencing volume was 10 l,
then for each sample, add:
2.5 l 125mM EDTA
30 l 100% EtOH
If your sequencing volume was 20 l then double the volumes above
2) Add 32.5 l of EtOH/EDTA solution to 10l of cycle sequencing product
3) Seal tubes and invert a few times to mix
4) Leave at room temperature up to 15 minutes to precipitate extension products.
5) Spin in refrigerated centrifuge 2500g for 30 minutes at 4oC (program #1 on Eppendorf
refrigerated centrifuges). Be sure to balance racks and tubes (Same number of tubes in each
rack (balancing tubes can be empty)):
IMPORTANT: proceed to next step immediately. If this is not possible, spin the tubes for 2
minutes before the next step
6) Remove seal and invert tray onto paper towel. Secure 2-3 paper towels over tubes with
rubber bands.
7) Place tray inverted into centrifuge and spin 50 g (up to 185 g) for 3 minutes (program #2).
8) Add 30l 70% EtOH to each pellet
ABI recommends making a fresh stock of 70% each time you do this. For each sample, add
21 l non-denatured 100% ethanol
9 l Water
9) Seal tubes and invert a few times to mix.
10) Spin plates 2000-3000 g for 15 minutes at 4oC (program #3).
11) Repeat steps 6 and 7 to remove 70% EtOH
Optional: some people put tubes in 65oC oven for 10 minutes to ensure EtOH evaporates
completely
Can also speed vac for 15 minutes to ensure evaporation
12) Samples are ready to be resuspended for 3730 run. They can also be covered in aluminum
foil and stored at 4oC
13) To run on 3730, add 10l Hi-Di formamide to each tube.
14) Heat for 5 min at 95oC and put on ice 5 min. NOTE: many people skip this step.
NOTE: the heating and cooling step here can be eliminated and you can load once the samples
are in formamide.
NOTE: Vacuum drying of samples not necessary
METHOD 2: Use the exact same protocol as above except steps 1 and 2 are as follows:
1) Make a master mix of EtOH/EDTA solution. If your final sequencing volume was 10 l ,
then for each sample, add:
1 l 125mM EDTA
1 l 3M NaOAc
25 l 100% EtOH
If your sequencing volume was 20 l then double the volumes above
2) Add 27 l of EtOH/EDTA solution to 10l of cycle sequencing product
Additionally, this protocol calls for 35 l of 70% (vs. 30 l as above)