Genotyping

Fragment analysis request form



The 3730 DNA analyzer is a 48-capillary instrument capable of performing DNA sequencing analysis, fragment analysis ( formerly called GeneScan ) and SNP analysis.

We are phasing out 377 gel based fragment analysis and switching to 3730 capillary system!

The main advantage of 3730 over conventional gel based instrument (377):



3730 capillary 377 gel
sensitivity Very sensitive, at least 10 times more sensitive than 377, therefore can significantly reduce the amount of template DNA used to do PCR. Sensitive
Sample loading Sample loading is automatic, reducing human errors, gel leaking, gel distortion etc. Manual gel preparation and sample loading. Prone to human errors and gel related problem.
how much to load? as a starting point, 1/10 of the amount for 377 gel. 1 relative to 3730.
Run speed 96 samples in one hour 96 samples in 6 hours
analysis Data automatic extracted, analysis is much easier with GeneMapper software Manually track gel, more time needed to analyze.

Submitting samples for fragment analysis on 3730.

         

please use 96 well PCR plate to submit samples ( we use PCR plate from GENEMate). Check with napcore staff about compatibility of PCR plate with 3730 instrument. You also need to submit your sample sheet as a microsoft excel file.  Use one of the two template files GS_48_template.xls or GS_96_template.xls to create your sample sheet.

3730 loads and run the first 48 samples from odd numbered columns (pink colored), it then loads and run samples from even numbered columns (white background). See the illustration below:


A01

A02

A03

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A12

B01

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C01

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D01

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G01

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H01

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If you have 1 - 48 samples and you want to load and run all samples in one run, you have to arrange your samples in all the pink colored wells. You also need to use the GS_48_template.xls file to do your sample sheet and input sample name in the well position that is used. Check with napcore staff on how to create your sample sheet. For 1- 48 samples arranged such, the instrument only load and run once and we charge $48 (external charge: $48)

If you have more than 48 samples (up to 96 in one tray), you can place your samples whatever way you want. But you need to use GS_96_template.xls to do your sample sheet and type in sample name in the well position that is used. Consult with napcore staff on how to create your sample sheet.The instrument has to load and run twice to run all samples and we charge $80 (external charge: $96)

If you have 1 - 48 samples and you arrange your samples in both pink colored and white colored columns, you need to use GS_96_template.xls to do your sample sheet, the instrument has to load and run twice to load all samples and we will charge $80 (external charge: $96)


3730 uses G5 filter set:


Color

Dye

Absorption Max (nm)

Emission Max (nm)

Blue

6-FAM

494

522

Green

VIC

538

554

Yellow

NED

546

575

Red

PET

558

595

Orange

LIZ

638

655

You can use 4 dyes to label PCR fragments and LIZ is used for size std.

We provide the following size standards:
LIZ 500 (orange)
ROX 500 (red)
ROX HD 400 (red)

LIZ 500 is the default size standard, but you can still use ROX 500 or ROX HD 400 as size standard if you don't use PET label.

The instrument can still run HEX, TET or JOE labeled fragment. But it can not run these dyes together with VIC and there may be some spectral overlap which should not cause any problem if you know what you are doing.



Data generated on 3730 is spectrally corrected and can be analyzed on Mac platform or MS PC platform. To analyze on Mac using GeneScan 3.0 and Genotyper 2.0, load data in GeneScan, open analysis setting and de-select multicomponent (if you select multicomponent, it will ask for a matrix file, data generated on 3730 don't need a matrix file). You also need to select which dye is used as size standard, to select red: hold down the control key and click on red column. You also need to make a size standard. After analysis using GeneScan, data analyzed can be imported to Genotyper program for further analysis.
We use GeneMapper 3.0 on MS PC to do analysis.



If you have started your project with 377 gel, it is better to finish your project with 377 gel unless absolute sizing is not a concern (such as LOH analysis). There might be sizing difference bewteen 377 gel and 3730 capillary. A fragment sized as 254 bp on 377 gel might be sized as 251 bp on 3730 capillary. But it will always be 254 bp on 377 gel and 251 bp on 3730 capillary.