01
INFOS AND TIPS
» Temporal Progress
» Ideal DNA Amounts and Concentrations
» Precipitation of DNA
» Four Hints for Plasmid Preparation
» Recommendations for PCR Products
» Information for Data Delivery
» Data and Compression Formats
» Software for Editing
» Advice for Sample Sending for postal Service
» Hints for postal Delivery
TEMPORAL PROGRESS
24h service, provided that there are no
problems with the samples, you will obtain sequencing results on the next day
after arrival of the samples at SEQLAB.
If the samples arrive before 11.30 am, cycle sequencing will be performed on the
same day, followed by overnight sequencing run.
The following day you will receive your results between 11.00 am and 6.00 pm.
Here are now the most important TIPS and
information to permit speedy delivery and
excellent sequencing results
PRECIPITATION OF DNA
ds-DNA
(bp)
best DNS
quantity (ng)
minimum DNAconcentration
ng/µl*
10000
2000
400
5000
1000
200
4000
800
160
3000
600
120
2000
400
80
1000
200
40
750
150
30
500
100
20
250
50
10
150
25
5
*lowest DNA concentration, higher concentrations are no problem, since
DNA can easily be diluted.
IDEAL DNA AMOUNTS AND CONCENTRATIONS FOR PREMIUM SEQUENCING RESULTS
In this table you will find the quantities
and concentrations of DNA for Full Service
sequencing. For HotShots and AdvantageRead sequencing additional infos will be
shown in the appropriate sections on the
homepage.
For sequencing we need the threefold to
fourfold amount of the „best DNA quantity“, so that we have the chance to repeat
the sequencing reaction.
• DNA quantity for plasmids : 4-8 µg DNA
depending on the length of the vector
• The minimum concentration for plasmids
is 200 ng/µl; for longer plasmids (vector +
insert > 8000 bp) we need concentrations
between 300 - 400 ng/µl.
• The plasmids and PCR products should only be dissolved in 5-10 mM Tris/Cl, (NO
EDTA!), pH 8.5! For transport you can also
sent us the dried DNA pellet with a note
of the exact absolute amount of DNA.
Please precipitate your DNA (including
plasmid DNA from midipreps or maxipreps)
only at room temperature. Also, ethanol or
isopropanol should be at the same temperature.
At lower temperatures plenty of salt coprecipitate, which may inhibit cycle sequencing reaction.
The DNA should be washed with the same
volume of 70% ethanol like you perform
the precipitation; let the DNA pellet stand
5-10 min under the washing alcohol. We
recommend a second washing step.
02
INFOS AND TIPS
FOUR HINTS FOR PLASMID PREPARATION
1.) After culture growing and precipitation
of the bacterial cells the culture medium
must be removed completely from the cell
pellet, because some components of culture medium can inhibit the cycle sequencing reaction. Then wash the cells with
TRIS buffer (10mM, pH 8.0) and remove
the buffer completely, please. Subsequent
cell lysis should be accomplished according
to the instructions of your miniprep protocol.
4.) Another critical working step in the
plasmid or PCR product purification is washing the column with ethanolic solutions.
Displaced ethanol in the DNA solutions will
inhibit cyle sequencing reaction! Please
follow exactly the given times and rotation
speed of centrifugation or elongate the
centrifugation time slightly.
2.) Overloaded plasmid kit purification columns deliver high amount of DNA, but
this DNA gives only poor sequencing signals, or the cycle sequencing reaction fails
completely.
The following tests raise the probability of
good sequencing results with PCR products:
Our advice is to purify only 1.5-2 ml of culture on a miniprep column, 20 ml on a midiprep column and 100 ml on a maxi prep
column!
3.) For Low Copy plasmids like expression
vectors, we suggest an alternative two
step purification procedure:
Prepare a larger volume of bacterial culture than described above and purify the
DNA on a first column.
Take only 10 –15 µg of DNA and purify the
DNA on a second miniprep column. Please
follow the instructions of your miniprep
protocol exactly ( the same amount of lysis buffer addition, and waiting periods
etc... )
RECOMMENDATIONS FOR PCR PRODUCTS
1) PCR products should be well reamplifiable. If the reamplification comes up with
only a faint band this will also lead to bad
sequencing results.
2) Make sure that your PCR product can t
be produced from one primer alone, so
called one-primer-PCR. For a test include
PCR reactions with forward and reverse
primer only. Any upcoming fragment reveals the corresponding primer to serve as
forward and reverse primer at the same
time. Any sequencing of such a PCR product will undoubtedly result in useless
double signalling.
This one-primer-PCR phenomenon is often
seen with genomic DNA templates.
3) PCR products should be well separated
from remaining primers and dNTPs, If PCR
products show a single DNA band in a control gel they should only be purified on a
purification column. Agarose gel purified
PCR products seem to degrade more easily
or faster.
Please dilute PCR products only in 5-10
mM Tris/Cl, pH 8.5 (NO EDTA!).
03
INFOS AND TIPS
INFORMATION FOR DATA DELIVERY
The sequence data, electropherogram (=
ABI trace file) and sequence text file, are
posted to your personal area on our server.
You will receive an e-mail message when
your results are available.
For delivery by postal services there is an
extra fee, while e-mails are free. If you
need other delivery formats (print, CDROM, disk or fax), you will find the prices
for these in our price list. Please tick off on
the order sheet which kind of data form
you wish.
DATA AND COMPRESSION FORMATS
The sequence data, ABI trace file with the
extension *.ab1 and sequence text file
with the extension *.seq, will be compressed to a zip archive and then placed to
your disposal on our server.
PC user will receive data as a zip archive
attached to an e-mail. Mac user can choose
between three compression formats: zip,
sit or sea. Data delivery on disk or CD-ROM
will mostly be not compressed, but for larger amounts of data you can select between the mentioned formats.
Sea files are selfextracting on Mac computers, PC owner can use the program Stuffit
for decompression.
You will find Stuffit for download on
the web:
www.softwaretocompress.com
SOFTWARE FOR EDITING
For viewing and editing of electropherograms, the following programs
exist which can be downloaded for
free:
for PCs
• CHROMAS HYPERLINK „http://www.
technelysium.com.au/chromas.html“
• Finch TV
http://www.geospiza.com/finchtv/
for MACs
• EDIT VIEW from Applied Biosystems
http://www.appliedbiosystems.com/
support/software/
for older MacOS versions up to 9.2
• Finch TV
http://www.geospiza.com/finchtv/
also for Mac OSX
HINTS FOR POSTAGE DELIVERY
Deliveries of letters by the „Deutsche Post“
should be addressed to the post box (in
German: Postfach).
SEQLAB
Sequence Laboratories Göttingen GmbH
Postfach 3343
37023 Göttingen
If you use Express Services instead for the
sending of letters or small and big parcels
please address these with the following:
SEQLAB
Sequence Laboratories Göttingen GmbH
Hannah-Vogt-Straße 1
37085 Göttingen
0551 - 370 00 - 11
Dr. Werner Baussmerth
If you have any questions don‘t hesitate to
call me on this following number.
Or wright please an e-mail to:
[email protected]
ADVICE FOR SAMPLE SENDING FOR POSTAL
SERVICE
For transport purposes please use a shockprotected envelope and pack the sample
tubes additionally with pulp in a plastic
box, Falcon tube or little cardboard for
shipping. HOTSHOT samples, don‘t wrap
any parafilm etc. around the tube!