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Microsatellite Analysis Protocol

 

Materials:     

  • Buffer                           HiDi Formamide                                              ABI

  • Size Standard               GENESCAN 400HD [ROX]                        ABI

  • Sequencing Plate          Optical reaction plate [N801-0560]                  ABI

Safety Issues:

      Gloves& Lab coats should always be worn correctly

      Safety goggles must be worn when using reagents

      Read applicable MSDS sheets before handling reagents

      Specific fume cupboard regulations should be followed and the appropriate usage sheet signed

Hi-Di Formamide - may cause harm to the unborn child.

 

Procedure:

  1. PCR product should have been run on appropriate % gel to check purity and facilitate dilution factor calculations.
  1. Dilute PCR product according to intensity of band on gel.  Normally 1/101/50 dilution in ddH2O.
  1. Mix diluted samples by gently pipetting, cover plate with adhesive seal and spin samples briefly in Centaur 2 (Post-PCR) centrifuge.
  1. Create sufficient master mix of HiDi and ROX for all samples and aliquot 11 μl of master mix into each well of optical plate.

 

HiDi                 10.7 μl

ROX                  0.5 μl

                         Total per reaction      11.0 μl

  1. Add 1.0 μl of sample (diluted PCR product) into each well.  Cover plate with adhesive seal and spin briefly in Centaur 2 (Post-PCR) centrifuge.
  1. Denature DNA by heating plate to 90 oC for 2 minutes and immediately place on ice prior to loading on 3100 genetic analyser.
  1. Remove the adhesive seal and reseal the plate using a grey rubber lid.  Lids may be found below the 3100 genetic analyser, or removed from a completed plate.  Wash grey lid with mQ water and blot dry using tissue paper before sealing the plate.
  1. Create a new plate layout in the Data Collection software (on computer connected to 3100 genetic analyser).  The module and analysis runs depend upon which array is installed and with buffer is currently running in the machine.  Generally, the layout will be:

 

Sample Name             Depends on sample!

BioLims                      3100_Project1

Dye                             D

Run Module               GS36-POP6

Analysis Module        GS400HDAnalysis.gsp

 

Following 3100 runs, files should be saved to CD / Zip drive and the plate removed from the 3100 genetic analyser and disposed of in the appropriate yellow sharps bin in the left fume extraction cabinet in the QUB research lab.

  

Additional Information...

 Genescan module:                                              36cm array                 36cm array     50 cm array

POP 6                          POP 4              POP 6

 

                                    Run Temp                                   50                                60                    50

Capillary fill vol                         184                              184                  184

Pre run voltage                         12.2                             15                    12.2

Pre run time                              180                              180                  180

Inj volt                                      3.0                               3.0                   3.0

Inj time                                     10                                10                    10

Run volt                                    15                                15                    15

Data delay                                1                                  1                      1

Run Time                                  3000                            1500                4000

 

400HD Rox:      

  • Identifies fragments 50 400 bp in size with no anomalies like ROX 350 / 500

  • High density size standard for microsatellite analysis

  • Each fragment has ROX fluorophore

  • Recommended for ABI linkage mapping sets

  

Peaks at (bp):       50                    60                    90                    100                  120                  150

                        160                  180                  190                  200                  220                  240

                        260                  280                  290                  300                  320                  340

                        360                  380                  400