Ready-to-Run Seq

Ready-to-Run Sequencing protocol

Customer provides:

Ready made sequencing reactions (10µl) preferably in a 96-well plate format.
Completely filled Sample submission form as e-mail attachment sent to sequencing(at)ktl.fi.

Please fill plates by columns, click picture to enlarge.

Prepare template:

Poor template quality is the most common cause of sequencing problems. We assume that you use DNA of known concentration in PCR and purified templates in your sequencing reactions.

Check DNA concentration, e.g. with NanoDrop ND-1000.
Verify success of PCR with agarose gel elctrophoresis.
Purify sequencing template (e.g. ExoI-SAP, EXO-SAPiT).

See Prepare Templates for more information.

Sequencing reaction setup:

Prepare a sequencing master mix (see table below).
Deliver 8.0 µl of master mix to each well.
Add 2.0 µl of purified template to each well.
Seal plate and spin samples down.

NOTE: Fill plates by columns: A1, B1, C1..
>> see help

Applied Biosystems recommends following amounts of template for sequencing:

Template length	Quantity
PCR 100-200 bp	1-3 ng
PCR 200-500 bp	3-10 ng
PCR 500-1000 bp	5-20 ng
PCR 1000-2000 bp	10-40 ng
PCR >2000 bp	20-50 ng
ssDNA	25-50 ng
Plasmid DNA	150-300 ng

(from BigDye Terminator v3.1 Cycle Sequencing Kit protocol)

BigDye dilutions for different templates.

Template length [bp]		PCR less than 200 bp	PCR 200-500 bp	PCR 500 - 800 bp	over 800 bp (plasmid)
Dilution ratio	[c]	1:50	1:32	1:16	1:12
Milli-Q Water		5,30 µl	5,20 µl	5,10 µl	5,00 µl
BigDye Dilution buffer v3.1	5X	1,90 µl	1,90 µl	1,75 µl	1,65 µl
BigDye RR mix v3.1		0,15 µl	0,25 µl	0,50 µl	0,7 µl
Your sequencing primer	5µM	0,65 µl	0,65 µl	0,65 µl	0,65 µl
Template (purified)		2,00 µl	2,00 µl	2,00 µl	2,00 µl
Total rxn volume		10 µl	10 µl	10 µl	10 µl

NOTE: Dilutions have only been tested for BigDye v3.1 chemistry with original Dilution buffer from Applied Biosystems.

Seal the plate properly with PCR tape.
Spin samples down with centrifuge – SeqLab plate centrifuge is at your service.
Proceed to thermocycling

Thermocycling

Place your samples in thermocycler and run following cycle sequencing protocol. SeqLab recommends the modified protocol to gain stronger signals.

Original BigDye 3.1 protocol (Applied Biosystems)

96°C for 1 min

Initial denaturation

24 cycles of

96°C for 10 s
50°C* for 5 s
60°C for 4 min

Use rapid ramp (1°C/s)

Denaturation
Primer annealing (50 - 60°C) *
Extension

10°C forever

Hold until delivered to SeqLab

Modified BigDye 3.1 protocol (used for Full Service sequencing)

96°C for 1 min

Initial denaturation

29 cycles of

96°C for 10 s
50°C* for 10 s
60°C for 4 min

Use rapid ramp (1°C/s)

Denaturation
Primer annealing (50 - 60°C) *
Extension

10°C forever

Hold until delivered to SeqLab

NOTE: You can modify primer annealing temperature* according to your primer (max. 60°C)
NOTE: If the samples are evaporated you can try increase volume to 10 µl with Milli-Q water.

Submit order & Deliver samples

Are you sending samples by mail outside Biomedicum? See "Drying the Samples".
Spin samples down with centrifuge – SeqLab plate centrifuge is at your service.
Submit your order (See instructions).
- Download and fill Sample submission form completely.
- E-mail Sample submission form as an attachment to sequencing(at)ktl.fi.
Deliver samples to SeqLab, see Deliver Samples.
You will be informed by e-mail when results are stored to your account at SeqLab's database.
Proceed to Getting Results.

Questions or problems regarding this web site should be directed to SeqLab.
Last modified: 09/10/08.