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The Center for Genomic Technologies : MIcrosatelite genotyping

The Technology:
Microsatellites are polymorphic loci present in DNA that consist of repeating units of 1-6 base pairs in length. They are used as molecular markers which have wide-ranging applications in genetics including linkage, association studies and forensic.
 
Microsatellites can be amplified for identification by the polymerase chain reaction (PCR) process, using the unique sequences of flanking regions as primers. The use of one primer labeled with a fluorescent dye, and the other unlabeled primer in the PCR reaction results in a labeled PCR product that is detected and sized by the ABI PRISM™ 3730xl DNA Analyzer. Collected data is analyzed by the GeneMapper or Peak Scanner Software.
  
Software:
GeneMapper® Software
can be purchased through the local agent Rhenium:
Phone: +972-8-9558888
   
Peak Scanner ® Software can be free downloaded
 
For WINDOWS XP
 
For WINDOWS 7 download software "Peak Scanner™ 2" from this link:http://resource.lifetechnologies.com/pages/WE28396/
 
Dye sets:
The two dye sets for detection and sizing labeled PCR product is available in the Center:
DS-30: Size standard ROX-400. PCR product should be <400bp and labeled with 6-FAMTM, HEXTM, NEDTM.
DS-33: Size standard LIZ-500. PCR product should be <500bp and labeled with 6-FAMTM, VICTM, NEDTM, PETTM.
 
Sample submission:

1. PCR are performed by customer

Labeled PCR amplification products are submitted. PCR products should arrive in a 96-wells plate even if the plate is not full. The order of the samples in the plate should always be as follows:
Sample 1 is in position A1
Sample 2 is in position B1
………………………….
Sample 96 is in position H12
 
The samples should be diluted appropriately before submission.    
Table with names of the samples should be send by E-mail.
 

2. PCR are performed at the Center

  • with primers provided by the customer
  • with primers available at the Center (see Table-Markers)
DNA samples are submitted. Concentration of DNA >100ng/µl. The samples should arrive in 1.5ml tubes. The concentration of customer’s primers should exceed 10µM and be noted on the tube.

3. Whole Genome Scan with 400 microsatellite markers (linkage associated studies)

ABI linkage mapping set MD10, of about 10cM distances is available at the Center. Other linkage mapping sets (LD-20 and HD-5) or special markers are also available upon request. Genomic DNA samples are submitted. Concentration of DNA >100ng/µl. Total amount 20 µg. The samples should arrive in 1.5ml tubes.

The Center for Genomic Technologies

 

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Faculty of Science The Hebrew University of Jerusalem The Hebrew University of Jerusalem