CBS DNA Sequencing Protocols
Set up PCR run
BigDye Terminator v3.1 Labeling Reaction
Big Dye
5X SeqBuffer
Primer
DNA
Water
DMSO
FINAL VOL.
10 pmol
2.5ng/100base
5%
1 ul
1 ul
1 ul
10 ul
optional
15 ul
1. Calculate the amount of primer to use in PCR reaction
It is recommended to add 1 ul of 10 pmol/ul primer or 2 ul of 5pmol/ul primer. If your
primer concentration is 2 pmol/ul, it is recommended to add 2.5 ul of primer.
2. Calculate the amount of DNA to use in PCR reaction
Use 2.5 ng /100 bases of total DNA to calculate the amount of DNA you should use.
3. Make Master mix with BigDye: buffer: water =1:1:10
Always make an extra to make sure there is enough for all the samples. For example,
if you have 10 samples, you could add 11 ul of BigDye, 11ul Buffer and 110ul water.
Mix well and then add 12 ul to each PCR tube.
4. After adding the master mix, add the calculated amount of Primer and then DNA
template to each PCR tube. Mix well and spin for 30 sec ensure samples are at the
bottom of each well.
5. Refer the following to set up running condition.
PCR Cycling Conditions
1.
960C for 2 min
2 Step PCR Cycling Conditions
1.
960C for 2 min
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February 12, 2016
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2.
3.
4.
5.
6.
7.
960C for 30 sec.
500C (450C or 550C)
for 15 sec.
600C for 4 min
Goto step 2, 29X
Hold at 40C forever
End.
2.
3.
960C for 30 sec.
600C for 4 min
4.
5.
6.
Goto step 2, 29X
Hold at 40C forever
End.
Preparing the Sephadex plates
Make Sephadex plates using column loader and then hydrate with 300ul water. Wait
for 3 hours with Sephadex plate sit on counter or one hour when removed from fridge
before using it. Sephadex plates can be stored in fridge for a few days.
Post PCR Reaction
1. Briefly centrifuge DNA sequencing reaction plate or tubes.
2. Carefully load 15 ul of the sequencing reactions to the center of each Sephadex
column.
3. Place the AcroPrep 96 Filter Plate (containing the sequencing reactions) with the Pall
Alignment collar on top of a clean AB3730 compatible 96 well reaction plate
containing 10 ul of Hi-Di Formamide in each well, and centrifuge for 3 minutes at
750g.
4. Seal plate with septum .
5. Place the PCR plate with septum into black base plate and secure assembly with the
plate retainer. Ensure that the holes of the plate retainer and the septa are aligned.
6. Load plate assembly into AB 3730 DNA Analyzer and Import plate record.
7. Before initiating sequencing, ensure that Oven door, Instrument door and Stacker Drawer
are closed. Make sure that adequate levels of buffer and water are in the appropriate
reservoirs. Check the level of POP-7 polymer in the bottle to ensure sufficient volume
for run.
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February 12, 2016
ProtocolsCBS.doc