Obtaining the sequencing results
|About the Unit
For any query send e-mail to Tslil
Ophir or call 03-6406370
The DNA Sequencing Unit at the G.S. Wise Faculty of Life Sciences,
was established in 1996 as part of the Interdepartmental Equipment Unit.
It's aim is to provide sequencing services with the latest technologies to
researchers from Tel-Aviv University and to all other interested scientists.
Automated DNA Sequencing
Our method of sequencing utilizes the BigDye Terminator Cycle Sequencing
Kit from AB.
Here each of the Dideoxy Terminators has a fluorescent dye attached to it.
The reacted DNA fragments is separated and the fluorescence is detected.
Signals are sent to a computer and collected by ABI's Data Collection Software,
then interpreted by ABI's Analysis software..
Since July 2001 the Unit runs the ABI PRISM 3100 Genetic Analyzer as its main sequencing apparatus.
The ABI PRISM® 3100 Genetic Analyzer is a multi-color
fluorescence-based DNA analysis system using the proven
technology of capillary electrophoresis with 16 capillaries operating
in parallel. The 3100 Genetic Analyzer is fully automated from
sample loading to data analysis.
Sixteen capillaries provides the capability to analyze
hundreds of samples per day and increases laboratory
Automated polymer and sample loading and easy data
analysis provide an easy to use system with minimal user
intervention to reduce labor costs and to increases lab productivity.
Application flexibility provides both sequencing and
fragment analysis applications quickly, accurately, and reliably .
Multiple capillary lengths and polymer choices combined
with proven chemistry kits provide a complete system with
the highest number of supported applications.
The ABI PRISM® 3100 Genetic Analyzer includes patented
technology licensed from Hitachi, Ltd. as part of a strategic
partnership between Applied Biosystems and Hitachi, Ltd., as well
as patented technology of Applied Biosystems.
For Research Use Only. Not for use in diagnostic procedures. .
(From the AppliedBiosystems site)
For gel separations we use a 4.8% 48cm well-to-read acrylamide gels on a ABI PRISM 377XL sequencer, which enable us to read up to 700bp from a properly prepared template DNA. The data is then interpreted by ABI's Analysis software.
BigDye Terminator Specifications
ABI PRISM sequencing kits provide all of the reagents and protocols
needed for sequencing 100, 1000 or 5000 single-stranded or double-stranded
DNA templates. The minimum performance specification using the pGEM control
and the -21M13 primer are as follows for the ABI PRISM® 377 DNA Sequencer:
The reagents provided in each kit have been optimized for use with ABI PRISM 377 and 377XL DNA Sequencers, 3100 Genetic Analyzer, and GeneAmp® PCR systems.
Gels are also used for Fragment Analysis. Here labeled primers are used
in PCR amplification to generate fluorescent labeled fragments.
The fragments are then separated on a 5% 36 cm well-to-read Long Ranger (BMA) gel.
A size standard is used in each lane enabling a very accurate and reproducible size calling. Results are analyzed by AB GeneScan software.
What to bring ?
The following types of DNA are suitable for sequencing. Please provide the specified amount of DNA:
1) M13/pUC reverse primer (-48)
2) M13/pUC reverse primer (-21)
5' - AAC AGC TAT GAC CAT GAT TAC G - 3' 22 mer
3) M13/pUC uviversal primer
5' - TGT AAA ACG ACG GCC AGT - 3' 18 mer
4) M13/pUC universal primer
5' - GTT TTC CCA GTC ACG ACG TTG - 3' 21 mer
5) T3 primer
5' - GCG CGA AAT TAA CCC TCA CTA AAG - 3' 24 mer
5' - AAT TAA CCC TCA CTA AAG - 3' 18 mer
6) T7 primer
5' - CGC GCG TAA TAC GAC TCA CTA TAG - 3' 24 mer
5' - TTA ATA CGA CTC ACT ATA GG - 3' 20 mer
7) GT 10 forward
5' - GAG CAA GTT CAG CCT GGT TAA G - 3' 22 mer
8) GT 10 reverse
5' - GGC TTA TGA GTA TTT CTT CCA GGG - 3' 24 mer
5' - CGA GGT CGA CGG TAT CG - 3' 17 mer
5' - TCT AGA ACT AGT GGA TC - 3' 17 mer
5' - CCA AGC TAT TTA GGT GAC AC - 3' 20 mer
12) SP6A (for
5' - GCA TTT AGG TGA CAC TAT AG - 3' 20 mer
5' -TAG GGG AAT TGT GAG CGG ATA AC - 3' 23 mer
14) T7 Terminator
5' - GCT AGT TAT TGC TCA GCG GT - 3' 20 mer
15) pGEX 5'
5' - GGG CTG GCA AGC CAC GTT TGG TG - 3' 23 mer
16) pGEX 3'
5' - CCG GGA GCT GCA TGT GTC AGA GG - 3' 23 mer
17) POLY-T (for
sequencing down stream poly-T regions)
5' - TTT TTT TTT TTT TTT TTT TTT TV - 3' 23 mer
18) POLY-A (for
sequencing down stream poly-A regions)
5' - AAA AAA AAA AAA AAA AAA AAA AB - 3' 23 mer
Viewers for the chromatogram files
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