Microsatellite Genotyping

 

DNA plates:

 

--Plate design: arrange controls in center of plate, in diagonal line, to

maximize numbers of rows and columns that contain a control-

-controls include parents and/or grandparents, repeats, blanks, known samples

 

                  --Main stock plate: dilute all DNAs to 0.05 mg/ml in nuclease-free TE

(50 ml/well, store at -80°)

 

--Working stock plate: dilute 5 ml of each DNA in above plate with 45 ml

nuclease-free 10 mM Tris, pH 8--final concentration 5 ng/ml (use 2.5 ng/reaction)

 

Primers:

 

--Primer purchase: obtain red, green, and yellow-labeled forward primers from

ABI--obtain blue forward primers and unlabeled reverse primers from IDT

                 

--Main stock tubes: resuspend primers in nuclease-free 10 mM Tris, pH 8, to a

concentration of 100 mM (store at -20°)

 

                  --Working stock tubes: dilute stock 1:10 in nuclease-free pure water (to 10 mM)

arrange primers for each day in Sarita's "Primers" box at -20°

 

PCR:

 

                  --PCR reaction:  See attached PCR Worksheet (for 384-well plate)

                    

       For each reaction (5 ml total):

 

                                    --0.5 ml forward primer )

                                    --0.5 ml reverse primer  ) combine these in primer mix (2.5 ml/reaction)

                                    --1.5 ml pure water        )

 

                                    --0.5 ml DNA from working stock plate

 

                                    --0.5 ml 10X PCR buffer      )

                                    --0.3 ml 25 mM MgCl2        )

                                    --0.0625 ml 20 mM dNTPs   ) combine these in PCR mix (2 ml/reaction)

                                    --0.025 ml AmpliTaq            )

                                    --1.1125 ml pure water         )

 

                 

Date: _________

 

Genotyping: PCR Worksheet

 

Note: Use filter tips throughout

 

Primers:  Primer name (check same for F,R):  __________

 

10uM forward primer: 225 ul                          _________

                                         10uM reverse primer:  225 ul                          _________

                                         MilliQ water:                      675 ul                          _________

 

                           Mix well and aliquot 69 ul into 16 wells:           _________

 

                           Then dispense 2.5 ul into each well (x384):     _________

 

DNAs:   Pipet 0.5 ul each DNA into appropriate well:      _________

 

PCR Mix:               10X PCR Buffer II:        225 ul                          _________

                                         25 mM MgCl2:                  135 ul                          _________

                                         20 mM dNTPs:                  28.1 ul                        _________

                                         Taq polymerase:              11.25 ul                     _________

                                         MilliQ water:                      500.6 ul                     _________

 

                           Mix well and aliquot 55 ul into 16 wells:           _________

 

                           Then dispense 2 ul into each well and mix:      _________

                           Spin down plate in centrifuge

 

PCR Program: KT-MS--any changes?  ________________

 

Notes:

 

--PCR program:  KT-MS:

 

95šC      1min 45s

56šC      45s

72šC      45s

 

94šC      45s          )

56šC      45s          }   x       4 cycles

72šC      45s          )

 

90šC      45s          )

56šC      45s          }   x       29 cycles

72šC      45s          )

 

72šC      5min

4šC         hold

 

3730 Plate Assembly:

 

                  --96-well plate:  Combine Genescan500-LIZ size standard and Hi-Di Formamide

                                    --27.5 ml Genescan LIZ (ABI # 4322682)

                                    --797.5 ml Hi Di Formamide (ABI # 4311320)

 

--Aliquot 103 ml into each tube of 8-tube strip, then use multichannel

pipettor to aliquot 7.5 ml into each well of 3730 plate

 

                                    --Add 0.7 ml of each PCR reaction into appropriate well--pool together red,

blue, green, and yellow primer reactions

 

                                    --Spin down plate briefly in centrifuge

 

--Denature plate at 95° for 2 minutes, then quickly place on ice for 10

minutes

 

                  --384-well plate: see worksheet on next page

 

Misc. 3730 info:

 

--The water and buffer (10X 3730 Buffer, ABI # 4335613) should be changed daily.  Dilute the 10X buffer stock 1:10 in pure water--make 150 ml for the buffer reservoir tray (80 ml) plus the buffer bottle (70 ml).  Remove the three trays and buffer bottle, empty and rinse with pure water, then refill with water or buffer

 

--The polymer blocks should be washed with water once a week--use the "Water Wash Wizard" and follow directions

 

--The polymer (POP7, ABI # 4335615) should be refilled as needed--store at 4°

 

 

Date: _________

Genotyping: 384-Well Plate Assembly for 3730

             

Note: Use unfiltered tips throughout

 

Markers: ______________________________________________

 

Get 384-well plate for 3730, bar code: _________________

             

Pipet MilliQ water into each well: 2 ul                _________

 

Add:  Blue PCR reaction:                     0.5 ul _________

                           Green PCR reaction:                  0.5 ul _________

                           Yellow PCR reaction:               0.5 ul _________

                           Red PCR reaction:                       0.5 ul _________

              Then spin down plate in centrifuge

 

Dehydrate samples in PCR machine, 80° for 10-20 minutes

--check every 5 minutes until dry

 

Meanwhile make Formamide/Standards Mix (in 5 ml tube):

 

                           GS500LIZ Size Standards:   125 ul            _________             

                           HD Formamide:               2375 ul         _________

 

              Mix well and aliquot 155 ul into 16 wells:        _________

 

              Then dispense 5 ul into each well and mix:      _________

              Spin down plate in centrifuge

 

Denature: Denature program on PCR machine, 95° for 2 minutes

                           --then place on ice for 10 minutes

 

Cover with septa and double-check alignment:         _________

Analysis of genotype data in GeneMapper:

 

--Open the GeneMapper program

 

--Under "File," select "Add Samples to Project"--this opens a new window where you can locate your files on the hard drives.  Select the file, then click "Add to List," below on the left (the file should appear in the right text box)--then click "Add and Analyze" below on the right.  The window will close and the program will prompt you for a name for your project--enter a name, and click OK

 

-  If the samples are not in the desired order, you can sort them by selecting "Sort" under the "Edit" menu--sort by sample file to organize by well position (A1, A2, etc.)

 

-  To see the plot for a given well, select it under "Sample File," then click on the red/blue/green striped button in the toolbar above "Samples" (or you can select "Display Plots" under the "Analysis" menu)

 

-  To enlarge particular regions of the plot, move the cursor near the axes, just outside the plot, until the cursor becomes a magnifying glass--then hold the mouse button down while dragging the cursor up or to the right to form a box.  The area inside the box will be enlarged.  This will only work for one axis at a time (enlarges either horizontally or vertically but not both)

 

    --To create a panel:

 

--Under Tools,  select Panel Manage--opens new window

 

--On the left screen, select a kit, or create a kit by selecting Panel Manager, then "New Kit" under File--name and select kit in left window

 

--Under File, select New Panel--name, then select in left window (may stall here)

 

--Under File, select New Marker--add marker name, color, and size range (suggest 10 bp below smallest allele and 10 bp above biggest allele)--click return after finishing each marker, then repeat New Marker to add additional markers

 

--Click Apply, then OK to exit window.

 

--On Samples sheet, under Panel, select new panel and fill down for all samples

 

--Click on green arrow on Samples page to analyze data and generate Genotypes files

 

    --To create bins (for automatic base calling):

 

--Return to Panel Manager window

 

--Make sure kit has bin set, or create new bin set under Bins

 

--Select panel in left window (may take few minutes)

 

--Under Bins, select Add Reference Data--select data set in new window, then click Add to List, then click Add

 

--Under Bins, select Auto Bin--change Minimum quality value to 0.08, and select "Rounded basepair"--then click OK

 

--In left window, click plus next to panel to view markers--select marker to view bins--red crosses indicate position of peaks in reference data set

 

--Click Apply to accept bins

 

--Bin adjustment: double-click on bin (gray column on plot) to open Edit Bin window--can change name, position, or size (suggest enlarging to 0.8 offsets)--can also click on bin and drag to move, or click on black box in center and drag to resize

 

--Can use blue buttons in top toolbar to add, delete, or edit bins

 

--When done with all markers, click Apply, then OK

 

--Click on green arrow on Samples page to re-analyze (make sure proper bin set is selected; see Analysis Method Editor below)

 

--Check through results on Genotypes page for errors--return to Panel Manager to refine bins, if needed (or change size range in panel to cut off stray peaks)

 

    --Other ways to optimize genotype analysis:

 

--Adjust settings in Analysis Method Editor--open menus by selecting a sample on Samples page, then selecting Analysis Method Editor under Analysis (or clicking blue bell curve button on top toolbar)

 

--Under Allele, make sure bin set selected matches bin set for kit

 

--Under Peak Detector, select Advanced--can adjust Peak Amplitude Thresholds to set cut-off for peak-calling--set polynomial degree to 5

 

--Under Peak Quality, can reduce Heterozygous balance (to 0.001, e.g.)

 

--For samples with failing Size Quality scores (SQ on Samples Page), you can (sometimes) override by selecting the sample, then clicking the red button on the toolbar to open the Size Match Editor--click Override SQ, then click somewhere inside the graph, then click apply and OK--click the green arrow to re-analyze

    --To export GeneMapper data:

 

--Switch table setting to Export format (in top toolbar; see below).  Sort samples as desired, then select Export Table under File--select location to save table, then export as Tab-delimited text (.txt)

 

--Table settings can be altered in Table Setting Editor, under Tools--to create a new Table Setting, select GeneMapper Manager, under Tools--then select Table Settings and click New

 

--To get program to list single peak as two homozygous alleles, select Options under Tools--under Analysis, click Duplicate homozygous alleles