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Keck
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DNA Sequencing >
FAQ
Frequently Asked Questions
Below are some of the more frequently
asked questions regarding sequencing reactions and data return. We
hope this will be useful for day-to-day questions. If you do not find
the answer here, please browse our information pages or call
203-737-2566
or
e-mail dnasequencing@yale.edu.
Why does my sequence file contain NNNN?
There is an insufficient level of termination products for the computer
software to call bases. Click HERE for a detailed explanation of
causes of "No analyzed data" results.
OTHER FAQs:
TEMPLATE
FAQ
PRIMER FAQ
TURNAROUND
FAQ
DATA RETURN FAQ
PRIMER WALKING
FAQ
HIGH VOLUME 96-WELL PLATE FAQ
TEMPLATE
FAQ:
How much plasmid DNA do you need for
a primer walking reaction?
We require 1 µg of template for
each sequencing reaction (i.e.primer/template combination). This is
three times the amount we use for each reaction, giving us sufficient
material for repeats if we have a technical failure. Template must be
provided at between 100-300
ng/µl.
How much plasmid DNA do you need for a
standard
premixed
tube reaction?
We require 600
ng of template for each sequencing
reaction. This amount is mixed with 2
µl of 4 µM primer and brought
up to 18 µl total volume
with H2O.
How much plasmid DNA do you need for a
high volume
premixed
96-well reaction?
We require 600
ng of template for each sequencing
reaction. This amount is mixed with 2
µl of 4 µM primer and brought
up to 18 µl
total volume
with H2O.
How much PCR product do you need for a
primer walking reaction?
We require
5-10 ng of
PCR product for every 200 bp of
product per reaction. Please supply
three times the required amount to cover technical failures.
How much PCR product do you need for a
standard
premixed
tube reaction?
We require 10-20
ng of PCR product
for every 200 bp of
product per reaction. This amount is
mixed with 2 µl of 4 µM
primer and brought up to
18 µl total volume with
H2O.
How much PCR product do you need for a
high volume
premixed
96-well reaction?
We require 5-10
ng of PCR product
for every 200 bp of
product per reaction. This amount is
mixed with 2 µl of 4 µM
primer and brought up to
18 µl
total volume with
H2O.
Should I resuspend template in TE or
H2O?
Either is fine. H2O is perhaps
better.
How long is the average read?
Template quality will affect length of read. Average read length
is 500 bp, but could be as much as 650-750 bp.
PRIMER FAQ:
At what molarity should primers be
submitted for primer walks?
All primers should be at 4
µM.
What size primers should I design?
Primers should be between 18-28 bases long.
What Tm value should primers
have?
Primers should fall within the range of Tm
values 55-65.
TURNAROUND FAQ:
When will I get my sequence if I submit
samples in individual tubes?
The turnaround time for individual tubes is 1-3
days depending on if the sample needs to be repeated. Most
sequences are returned within 1-2 days from
when we receive the sample.
When will I get my sequence if I submit
samples in a 96-well plate?
The turnaround time on the high volume
sequencing will vary depending on our general level of submissions.
It will generally range from
1-3 days.
DATA RETURN
FAQ:
Who should I call to ask questions
about data return?
If you must call to ask about data please call
737-2566.
Please leave a message if no one is able to answer the phone.
We will get back to you ASAP. You may
also leave an e-mail message at dnasequencing@yale.edu
What is your preferred method of data
return?
Data return via the
ftp-server
is our only method. A password protected ftp-account will be set up
for you and the ID and password (if a new account) will be e-mailed
to you after we have received your samples and submission
sheet.
If I lose a sequence can I have the data
re-sent?
We try to accommodate peoples' requests,
although you are responsible for your
data once it leaves our lab.
Retrieving data older than four weeks is time consuming. We reserve
the right to add an additional "data retrieval" charge for repeated
requests for old data. Please back-up
your sequence data right away to avoid this
problem.
When will I get my electronic data?
Electronic data is sent out daily between 1-4 pm, depending on the demands
of the lab each day.
PRIMER
WALKING FAQ:
Do you store primers and templates?
Once a project is completed the templates are discarded and
primers are returned to you. If you need the primers for another
project they must be resuspended at 4µM in microfuge tubes and
resubmitted.
What happens to the primers that were
designed for the project?
For Yale researchers -the remaining
primers are dried down and placed in an envelope, together with the
oligo specification sheet, and placed in the data return boxes in
BCMM 305 to await your pick-up. For outside investigators -the dried
primers are mailed out at the end of the project.
HIGH VOLUME 96-WELL
PLATE FAQ:
Who can submit samples in 96-well
format?
Anyone who has used our premixed sequencing format and is
confident that their DNA prep methods work for automated sequencing.
The whole plate is sequenced, and samples are run
on the ABI 3730xl capillary instrument.
WE CHARGE FOR EVERY WELL USED IN THE
96-WELL PLATE FORMAT REGARDLESS OF THE
RESULTS, unless there is a clear
technical failure on our part.
Why do I need to use the Red
Eppendorf Twin Tec or Applied Biosystems Plates?
We have
incorporated robotics into our procedures, and we cannot program the robot for
every plate type. For your convenience, the Red Eppendorf Twin Tec plates
are available in the stockrooms.
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