Sequencing

There are two formats to submit DNA sequencing request and the prices are different:

1) The premixed format
2) The traditional format

1) The premixed:
You submit DNA template and primer premixed in a tube. (If the primer is a standard primer available from the Napcore, you don't have to add the primer)

For one sequencing rxn, mix the DNA and primer according to the table below:


DNA template DNA and primer
PCR product mix 10 - 100 ng DNA and 2 picomole primer (1ul of 2 uM) in a total of 9 ul.

10 ng per 100 bp.

 i.e. for 400 bp product, 40 ng DNA is needed for sequencing.

100 ng is needed for fragments > 1 kb.
plasmids/phagemids/ M13phage etc. mix 200 ng DNA and 2 picomole primer (1ul of 2 uM) in a total of 9 ul
large insert clones (size: 50 - 300 kb)
such as cosmid, BACs, PACs etc
The premixed format cannot be used to submit large insert clones. Please use the traditional format to submit such template.

You fill out the premixed DNA sequencing request form.

Upon receiving your samples, we will use your premixed sample directly in sequencing rxn.

It is our policy to rerun all reactions that don't work, this only applies to the same submission, it would be wise to provide twice as much DNA and primer as needed in case of rerun (18 ul instead of 9 ul).

2) The traditional:
You submit DNA template and primer in separate tubes. (the Napcore provides standard primers)

For one sequencing rxn, submit the following amount of DNA template and primer in separate tubes (if 2 or more rxns are run on the same template, please double or multiple the amount of template DNA): 

DNA template DNA Concentration Sequencing primer
PCR product, 10 ul or more 10 ng/ul or higher 5 ul @ 2 uM
plasmids/phagemids/ M13phage etc., 10 ul or more 100 ng/ul. 5 ul @ 2 uM
large insert clones, 10 ul or more
(size: 50 - 300 kb, such as cosmid, BACs, PACs etc)
price is different for such template
200 ng/ul or higher 5 ul @ 6 uM

You fill out the traditional DNA sequencing request form

Upon receiving your samples, we will run an agarose gel to check DNA concentration and based on the concentration to use optimal amount DNA to set up sequencing reactions.

It is our policy to rerun all reactions that don't work (this does not apply to samples that have low DNA concentration), this only applies to the same submission, it would be wise to provide twice as much DNA and primer as needed in case of rerun.

FYI: Steps involved in DNA Sequencing:
1): Isolate/purify the template DNA. We recommend Qiagen kit for DNA isolation/purification.
2): Check DNA concentration by agarose gel electrophoresis along with a standard. O.D. reading tends to give false high concentration.
3): Design sequencing primer; due to mobility shift, the 3' end should be at least 10 - 20 nt from the start point where accurate read is needed. Napcore provides standard primers for sequencing only.
4): Fill out the online DNA sequencing request form, print out the form and bring it to Napcore along with samples. In most cases we rerun all reactions that don't work, it would be wise to give twice as much DNA and primer as needed in case for rerun.
5): We use BigDye Terminator v3.1 Cycle Sequencing Kit from Applied Biosystems for sequencing. Sequencing products are analyzed on a 3730 DNA Analyzer from Applied Biosystem.

Here is a typical sequencing rxn set up for plasmid:

Template DNA:             200 ng  (this is for plasmid )
Sequencing primer:        2 picomole  ( 1 ul of 2 uM)
Water:                          q.s.        (quantity sufficient)
Sequencing mix:            2 ul
_________________________________________

Total volume:                10 ul

Standard cycle condition:
25 cycles of
96 oC for 10 s
55 oC for 5 s
60 oC for 4 min

After cycling, sequencing products are ethanol precipitated, dried briefly in a speed-vac, resuspended in Hi-Di formamide and loaded on the 3730 DNA analyzer for capillary electrophoresis and analysis.

6): Sequencing results are emailed to you in a zip file.