Buffer HiDi Formamide ABI
Standard GENESCAN® 400HD [ROX]
Plate Optical reaction plate [N801-0560] ABI
Gloves& Lab coats should always be worn
Safety goggles must be worn when using reagents
Read applicable MSDS sheets before handling
Specific fume cupboard regulations should be
followed and the appropriate usage sheet signed
Formamide - may cause harm to the unborn
- PCR product should have been run on
appropriate % gel to check purity and facilitate dilution factor calculations.
- Dilute PCR product according to
intensity of band on gel. Normally 1/10 → 1/50
dilution in ddH2O.
- Mix diluted samples by gently
pipetting, cover plate with adhesive seal and spin samples briefly in Centaur
2 (Post-PCR) centrifuge.
- Create sufficient master mix of HiDi
and ROX for all samples and aliquot 11 μl of
master mix into each well of optical plate.
Total per reaction 11.0 μl
- 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.
- Denature DNA by heating plate to 90
oC for 2 minutes and immediately place on ice prior to loading on
3100 genetic analyser.
- 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.
- 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:
Depends on sample!
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.
36cm array 36cm array 50 cm array
POP 6 POP
4 POP 6
50 60 50
Capillary fill vol
184 184 184
Pre run voltage
12.2 15 12.2
Pre run time 180
3.0 3.0 3.0
10 10 10
15 15 15
1 1 1
Run Time 3000
fragments 50 – 400 bp in size with no anomalies like ROX 350 / 500
density size standard for microsatellite analysis
fragment has ROX fluorophore
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