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Fragment analysis
Fragment Analysis will be performed on a daily basis within the possibilities of the sequencing reactions which have to be processed.
First time users
The peak height of your results depends on the concentration of your PCR product. The concentration of your PCR depends on the amplification percentage of your marker/primer. Ideally, you should obtain peak heights of 2000-4000. Due to large differences in the amounts of markers being used it is not possible to recommend one specific dilution concerning the ABI3730xl runs. Previous tests have shown that some amplified material needed to be diluted up to 200-fold, depending on the marker that was used. To help you to obtain the best results, we advise you to deliver half a plate (48 samples) with scaled up dilutions of known samples, amplified with different markers of your list. You should start with dilutions from 50x up to 200x of the amplified material and then work down. When this first project has been discussed and approved by the LGTC staff, the costs for this test plate will be carried by the LGTC. The LGTC will also provide a plate if necessary. The results of the test will be discussed with you.
Sample delivery
Samples should be ready-to-run for analyzing by the ABI 3730xl. Deliver them in a 96-well skirted plate (MicroAmp Optical 96-well Reaction Plate Applied Biosystems Part# 4306737 with barcode 128). You can also deliver your plate without barcode, we have barcode stickers. Leave your plate in the lower drawer of the small LGTC freezer in R-05-23. Mention your name (same as on the order report), date, and which size standard you have used in the plate. We will denature the samples by heating them for 2 minutes at 95 °C and spin them down before running them in the ABI 3730XL.
Please put your Order report in the dedicated tray on top of the freezer, lower drawer. No order report means no run.
Fragment analysis services
- 4 dye Fragment Analysis with ROX as size standard.
- 5 dye Fragment Analysis with LIZ as size standard.
- 384-well plates ROX and LIZ as size standard.
We accept full 96 well plates and half 96 well plates (48 samples), but be sure to fill the empty wells with MQ.
384-well plates can also be delivered. Contact us for the possibilities. Plates can be purchased via the LGTC (ABI Prism 384 Well Clear Optical Reaction Plates with Barcode [code 128] Part No. 4309849).
Cancellations
As long as your fragment run has not been placed in the ABI 3730XL you can cancel your order.
Sample sheets
If you have a sample sheet you can mail it as an attachment to info@lgtc.nl and mention "sample sheet fragment run" in the subject. Incorrect sample sheets will be replaced by a standard version.
Templates for ABI 373XL samples sheets for 96- well plates can be found here:
Ask the LGTC for 384-well plates sample sheet ROX or LIZ.
For Windows users: open the file in a spreadsheet (e.g. Excel) and edit the sample names, only edit the sample name "add_sample_name". Avoid using spaces and all other signs related to spreadsheets ()*/+=. etc.
Data
Fragment run results (fsa files) should be available for collection within 3 working days. However, we try to deliver your results the next day when you deliver your plate before 5 pm.
Data accessibility
The results will be accessible from a network drive. You need to be authorized by the LGTC to access this drive, so if you don't have it yet, please mention it on your order form or mail your request to info@lgtc.nl. We will keep your data only for one month so be sure to download your data and make your own backups.
For Windows users: this is how you install the link to the fragment run result network drive:
- On your desktop right-click and select "new shortcut" and paste in the following location in the item box: \\10.160.8.146\LGTC-3730-FragmentRuns
- Select 'next'
- Give the shortcut a name and select 'finish'
You can check this drive from your own PC within the LUMC once you have installed the link to it and have been authorized by the LGTC staff.
Linux or Mac users who want to deliver a sample sheet and install a link to the fragment run network drive can contact us for the possibilities.
Data analysis
The data analysis can be done with two software packages available at the LGTC, see software tools for more information.
- Gene Mapper, Applied Biosystems
- Gene Marker, Softgenetics
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