Introduction: Genei offers DNA sequencing service for Molecular biologists. Sequence
analysis using an automated DNA Sequencer uses fluorescent label dye terminators or
fluorescent label primers. We use ABI's AmpliTaq FS dye terminator cycle sequencing
chemistry which is based on Sanger's Sequencing method. Each run is termed as a 'Single
Pass Analysis' where the data is in the form of coloured peaks of light intensity known as
'Electrophoregrams'.
In automated DNA sequencing, fastidious preparation of template DNA
is crucial. Generally if the template DNA and primers are of high purity and under ideal
conditions, one can expect to read upto 400-500 bases, but initial 50-60 bases closer to
primer annealing region may not give good readable data. There might be cases where low
signal strength, high background, increased occurance of 'N' s and a short length of read
is seen. The major cause for all these may be attributed to insufficiently purified
template DNA.
Sequencing Service: Genei
now offers the following services
I)Single Pass Analysis (SPA) II)Primer walking of constructs (Sequencing of
cloned inserts)
I) Single Pass Analysis: Services offered for single stranded,
double stranded, lambda and PCR derived templates.
Service Provided:
SER 1A: SPA service to read upto 450-500 bases using one primer and template provided by
the scientist.
SER 1B: Genei offers special price for a single order having more than 30 samples.
Optional services provided :
SER 2: Further purification of template DNA by PEG precipitation.
SER 3: Column purification of PCR product using Qiaquick spin PCR purification system.
SER 5:
Plasmid isolation service: Includes growing of a cloned
E.coli colony with antibiotic
selection, extraction and purification of the plasmid DNA. Scientist can send their clone
as a plate/stab along with full information about the strain. Kindly note that presently
we can only extend this service to standard plasmids like pUC, pBluescript or pET
vectors.
Primer services provided :
SER 4:
Standard primer service: Includes routinely used vector primers M13 Forward and
Reverse, T7U,T3,SP6,KS and SK primers. For additional primers, refer DNA sequencing
primers in index. These primers are PAGE purified and tested for function in automated DNA
sequencing reactions.
SER 6: PAGE purification of crude primers supplied by scientist. Kindly send 20-30
µgs of crude primer for PAGE purification.
BGCO 4: Custom synthesis and desalting of primers required for sequencing.
SER 10: Designing of fresh primers required for sequencing service.
II) Primer walking of constructs : Primer walking of constructs (Sequencing of cloned
inserts) is an effective strategy to sequence the entire cloned DNA insert. Genei offers
to read upto 5kb insert, bidirectionally using this technique.
Initially, sequence data is obtained using a standard primer which hybridizes to the
vector sequence upstream of the insert DNA. Based on this initial data a custom
oligonucleotide is designed, synthesized and used to prime a second sequencing reaction.
The data obtained from the second reaction overlaps with initial data and extends the
sequence further downstream of the initial primer. By repeated cycles of custom
oligonucleotide synthesis and DNA sequencing the cloned insert is sequenced completely in
one direction.
For bi-directional sequencing, a second standard primer is used which binds the vector
in the other direction, the above process is then repeated and the complete sequence read for
both strands.
This service includes DNA extraction, purification, Primer designing, synthesis,
desalting, sequencing and assembling of the final sequence.
For convenience, we provide 8 primer walking services :
SER 22A: Primer walking of constructs upto 1 kb, complete read of one strand.
SER 22B: Primer walking of constructs upto 1 kb, complete read of both strands.
SER 23A: Primer walking of constructs upto 2.2 kb, complete read of one strand.
SER 23B: Primer walking of constructs upto 2.2 kb, complete read of both strands.
SER 8A: Primer walking of constrcts upto 3 kb, complete read of one strand.
SER 8B: Primer walking of constructs upto 3 kb, complete read of both strands.
SER 28A: Primer walking of constructs upto 5 kb, complete read of
one strand.
SER 28B: Primer walking of constructs upto 5 kb, complete read of
both strands.
How to process sample &
primers:
Send DNA samples along
with primers. Please note that sample DNA and primer samples should be of high quality -
refer note no. 1 (Sample Processing) & note no. 2 (Primer Processing). The sample
should be sent along with the Application for Automated DNA Sequencing Service.
Agarose gel analysis and / or spectrophotometric check of template DNA will be done for
all samples received free of cost. We will take up the sequencing service only if we are
satisfied with the quality of DNA. Please note that if we are not satisfied with the
quality / quantity of DNA samples, customer will be informed accordingly.
Note no. 1: Sample Processing: Genei recommends the use of protocol optimised
by ABI for DNA preparation. Protocol is available from Genei on request.
Template DNA supplied should satisfy the following criteria :
1.
Salt free
2.
RNA free and
3.
EDTA free
Template DNA must be finally resuspended in deionised water only.
Quantity (and concentration) of
template DNA required for SPA services:
Product
Concentration
(µg/ml)
Quantity (µgs)
ssDNA
250-500
1-2
dsDNA
250-500
1-2
PCR product
100-200
1-1.5
Note no. 2: Primer processing:
Criteria for primers used in sequencing:
Primer sequence selection,
synthesis and purification have significant effect on the quality of sequencing data. We
recommend the following criteria :
1.
High Purity (either
HPLC or PAGE purified).
2.
Primers 18-24 bases
long usually gives good specificity.
3.
Preferably 50 to 55%
GC content.
4.
No mismatch is
present and no alternative hybridization sites are present in the template.
5.
For cycle sequencing,
primer with melting temperature between 50 to 60°C is recommended. Avoid low melting
temperature (40 to 45°C.) Melting temperature (Tm) is calculated as 4 (G+C) + 2
(A+T).
6.
No secondary structure
particularly at the 3' end is present.
Quantity (and concentration) of primers to be provided for sequencing :
If primer needs PAGE purification,
scientist should send ~ 20-30µgs of crude primer.
How to send samples:
DNA must be transported in dry ice / coolant packs at sufficiently low temperature to
prevent degradation. DNA in solution should not be sent at room temperature.
DNA
(especially PCR product) can be sent as lyophilised sample. Care should be taken to see
that lyophilised DNA is not excessively dry as this leads to problem in reconstitution.
Customer is requested to send the Application Form duly filled in along with the samples
sent for Sequence Analysis.
Result of DNA Sequencing Service:
Genei will provide data of each sample in the form of (1) sequence (2) Electrophoregram (3) Floppy disk. On request, the data can also be sent by email.
Delivery time: SPA services: Approximately 2 weeks.
Primer walking services: Approximately 2 weeks for every 1 Kb.
Shipping Address:
Kindly ship the samples to the following address
Attention:
Ms. Janaki Babu, Bangalore Genei Pvt. Ltd.
No. 6, 6th Main, BDA Industrial Suburb, Near SRS Road, Peenya, Bangalore-560 058.
Ordering
Information
Cat #
Product
Size
Price
(Rs.)
DNA SEQUENCING SERVICES
Single Pass Analysis (SPA) Services for
single-stranded, double-stranded, lambda and PCR-derived DNA templates:
We offer SPA service using one primer provided by the scientist along with the
sample. To order custom made primer for
SPA from Genei, refer BGCO2.
The customer can also select from standard primer for SPA, refer SER 4 below.
SER 1 A
SPA service to read upto 450 - 500 bp (for order less than 30 samples)
Per Sample
1600
SER 1 B
SPA service to read upto 450 - 500 bp (for more than 30 samples as a
single order)
Per Sample
1500
Optional Services
SER 2
Sample Processing: PEG precipitation service
1 Sample
550
SER 3
Sample Processing: Purification of PCR product
1 Sample
650
SER 5
Sample Processing: Plasmid isolation service
(from E.coli colonies on a plate, Standard plasmids only)
per colony
950
SER 4
Primer Service: Standard primer service :(Select M13 forward or reverse, SP6, T3, T7 U, SK
or
KS)
per primer
400
SER 6
PAGE purification of crude primer supplied by customer
per Oligo
1200
SER 10
Primer designing required for sequencing service
per Oligo
500
Primer walking of constructs (Sequencing of cloned insert): Service includes DNA extraction, purification, primer designing, synthesis and
desalting, sequencing and assembly of the final sequence. We provide complete sequence
data in floppy disk and electrophoregram. On request, the data can also be sent by e-mail.
SER 22 A
Primer walking of constructs upto
1 kb (One strand)
per sample
7500
SER 22 B
Primer walking of constructs upto
1 kb (Both strands)
per sample
11000
SER 23 A
Primer walking of constructs upto 2.2 kb (One strand)
per sample
12500
SER 23 B
Primer walking of constructs upto 2.2 kb (Both strands)
per sample
20000
SER 8 A
Primer walking of constructs upto 3 kb (One strand)
per sample
18000
SER 8 B
Primer walking of constructs upto 3 kb (Both strands)
per sample
32000
SER 28 A
Primer walking of constructs upto 5 kb (one
strand)
per sample
29000
SER 28 B
Primer walking of constructs upto 5 kb (Both strands)
per sample
49000
Service
Tax @ 8% will be charged extra
Application for Automated DNA Sequencing
Service
(Photo copy of this form is to be filled in by the scientist and
sent along with the sample)
Order No. / Date
:
Name of the scientist
:
Institute / Address
:
SPA Services
DNA to be sequenced
: Plasmid, PCR Product or ssDNA
Total No. of Samples
:
Total Amount of DNA supplied
: _________ µg as Lyophilized /in
H2O/TE
Type of DNA Template
: GC rich / AT rich / secondary
structure long repeats /
homopolymer region / Other
Purification protocol used
: Alkaline lysis with PEG pptn/Column/
Resin/ Other (Specify the column
used for DNA purification)
Primer
to be sequenced with
:
M13FP / RP / T7U / T3 / SP6 / SK /
KS / others
If
primer supplied by scientist
Purified primer supplied (PAGE/HPLC)
: Yes / No
Sequence
of primer / length (This data is essential)
:
Quantity of
primers supplied
:
Annealing
Temperature used by scientist
:
Approximate size
of insert
:
Type of SPA Services
SER 1 A
SER 1 B
SER 2
SER 3
SER 5
SER 4
SER 6
SER 10
Number of Samples
Primer Walking Services
Total
number of samples
:
Sample sent as
: Purified
DNA / Stab / Plate
Growth
conditions
: Antibiotic used / Temperature
Size
of the plasmid
:
Approximate
size of insert
:
Type
of DNA Template
: GC rich / AT rich / secondary
structure / long repeats /
homopolymer region / Other
