Steps for Fragment Analysis
1.- Sample preparation:
Fragment Analysis Service is performed with groups of 16 samples, corresponding to 1 run of the ABI3130xl sequencer of 36 cm long 16 capillaries and POP7 polymer.
The Gene Scan Size Standard (by Life Technologies) is chosen by the user among those labeled with ROX (the most used size standard) or LIZ, and depending on the fragment length to be analyzed. The longest peak of the size standard should be longer than the labeled fragment with the longest size.
The mostly used dye to label the PCR product is 6-FAM (good performance and cheaper than others). However, our sequencer is calibrated to use all the following combinations:
- When Size std is ROX, eligible dyes are 6-FAM, VIC and NED.
- When Size std is ROX, eligible dyes are 6-FAM, HEX and NED.
- When Size std is LIZ, eligible dyes are 6-FAM, VIC, NED and PET.
The sample preparation protocol depends on the application (MLPA, t-RFLP, microsatellites, etc.). The following protocol is used by some of our users.
For a run of 16 samples (minimum number of loaded samples) a mix is prepared with
160.0 ul HiDi formamide + 6.4 ul Size Std = 166.4 ul mix
In each of the 16 wells of a run (for instance, the first and second columns of a 96 well plate), aliquot
10.0 ul previous mix + 1.0-1.2 ul PCR product (diluted 1/10 - 1/20)*.
(* the user should check the optimal PCR product dilution degree and mix proportions for his/her application).
Within each 16-sample run, it is recommended to include one or more positive controls and a negative one (PCR blank).
When restriction enzyme digestion is required, it will be carried out by the user.
2.- Sample delivery to the Core Facility:
PRBB users fill in and sign the Service Application and hand it together with the Fragment Analysis samples (usually, PCR product + size std + formamide) leaving them in the lab on the appropriate tray and fridge, respectively. External users may deliver samples and the filled in and signed Service Application by mail service. Any partially or incorrectly filled application can not be considered.
Samples will be delivered in plates by groups of 16 or multiples of 16, filling groups of two columns (corresponding to the 16 capillaries), 4, 6, 8, 10 or 12 columns.
We will perform thermal denaturation of the sample previous to loading into the sequencer.
When samples are loaded the same day or the day after their preparation, they may be preserved at 4 ºC. User should verity that the plate is properly sealed to avoid any liquid leaking during transportation. When a longer period before loading is expected, sample freezing is recommended. A single freeze/thaw cycle does not affect samples in a notable way.
3.- Results delivery:
PRBB users analyze the results with the GeneMapper software in the facility, using the lab computer controlling the 3130xl.
External users will decide analysis conditions and parameters, bin creation, etc. together with a facility staff member. In case GeneMapper software is not available at the user lab, our facility and the user will agree on how to deliver the data.