SEQUENCING UNIT

Obtaining the sequencing results



 
 
 
 
 
Mailing address:

Tslil Ophir

Sequencing Unit
Interdepartmental Equipment Unit 
Britannia Bldg. Room 16 
G.S. Wise Faculty of Life Sciences
Tel-Aviv University
Ramat-Aviv, Tel-Aviv 69 978
TAU map

About the Unit 

DNA Preparation

Primers

Viewers
 

 

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.

                                   Key Features

Sixteen capillaries provides the capability to analyze
hundreds of samples per day and increases laboratory
productivity.

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:
 
 
 
     Run
       Module
                 Specifications
 
      Seq
       Run 36E-2400
         450 bases at 98%
 
      Seq
       Run 36E-1200
         550 bases at 98%
 
      *Seq
       Run 48E-1200
         650 bases at 98%
 
* This module is usualy used by the Sequencing Unit.

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.

GeneScan

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:
Please notice:


Primers

Total volume of primers and sample should be 16ul

What not to bring!!!!

When to bring samples Obtaining the sequencing results

 
Universal Primers Provided by Sequencing Unit:

 
 
1)  M13/pUC reverse primer (-48)
    5' - AGC GGA TAA CAA TTT CAC ACA GGA - 3'   24 mer

 2) M13/pUC reverse primer (-21)
     5' - AAC AGC TAT GAC CAT GAT TAC G - 3'   22 mer

 3) M13/pUC uviversal primer (-20)
    5' - TGT AAA ACG ACG GCC AGT - 3'    18 mer

 4) M13/pUC universal primer (-40)
    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  primer
    5' - GAG CAA GTT CAG CCT GGT TAA G - 3'    22 mer

  8) GT 10   reverse  primer
     5' - GGC TTA TGA GTA TTT CTT CCA GGG - 3'    24 mer

  9) KS
      5' - CGA GGT CGA CGG TAT CG - 3'    17 mer

10) SK
      5' - TCT AGA ACT AGT GGA TC - 3'    17 mer

11) SP6
      5' - CCA AGC TAT TTA GGT GAC AC - 3'    20 mer

12) SP6A (for pcDNA3)
      5' - GCA TTT AGG TGA CAC TAT AG - 3'    20 mer

13)  pET
      5' -TAG GGG AAT TGT GAG CGG ATA AC - 3'    23 mer 

14) T7 Terminator (for pET)
      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



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