How to handle the AmpliGrid System Pipetting the master mix

How to handle the AmpliGrid System
Useful hints for set up and analysis.
The purpose of this quick start guide is to help first time
users to set up a successful experiment with the AmpliGrid
system.
Template Preparation
Single cells can be deposited on the AmpliGrid using different
techniques like:
• Flow cytometry (e.g. Beckman Coulter MoFlo™ - see Quick Start
Guides for the instruments)
• Micromanipulation (use 0.05x PBS to micromanipulate the cells)
• Laser microdissection
• Pipetting dilutions
If using a nucleic fluorescent staining, the deposition of the single
cells on the reaction site can be controlled either manually or by
using the Cell Detection software (OAX04117).
Extracted template material can be used for amplification on the
AmpliGrid.
• DNA can be dried on the AmpliGrid before adding the PCR
master mix
• RNA must not be dried on the AmpliGrid; always prepare a
second master mix solution incl. RNA
Extracted template material should be diluted in water; high
amounts of buffer with high salt concentration (especially PBS
or Tris) can inhibit the PCR reaction and can lead to bubble
formations in the master mix. Also residues of extractions / washing
can inhibit PCR reactions (e.g. from columns, phenol extraction).
Some extraction methods also contain ingredients which lead to
bubble formation which induces droplet bursting during PCR.
The concentration of the extracted nucleic acids should be used in
the range of 100pg human genomic DNA equivalent (e.g. as positive
control). For other sources of DNA and applications this may vary.
Primer
Primer can be either included into the PCR master mix or can
be dried on the AmpliGrid before adding the PCR master mix.
Primer can also be added if there is template material (cells or
DNA) deposited already. The decision whether the primer should
be dried or added to the master mix can be taken dependent on
the workflow. It is recommended to add the primer to the master
mix when only one primer pair (sense and antisense) is used and
dried on the AmpliGrid when different primer pairs should be
tested (take the advantage of only one master mix). Primer should
be dried on the AmpliGrid by using 1 µL of approx. 0.6 µM primer
(sense and antisense). It is not recommended to dry primers on
cells when doing RNA analysis since the drying process might
influence the quality of the RNA of the cells.
Productinformation AmpliGrid #PI10012 Version 1.0
Pipetting the master mix
• 1 µL of master mix should be pipetted on the AmpliGrid with
deposited template (and primer). Do not exceed that volume.
• We strongly recommend to use electronic multistep pipettes
(e.g. Eppendorf XStream™ or Rainin AutoRep™) to avoid the
creation of bubbles, which can lead to explosion of the reactions
(air bubble expands due to heating and explodes under the oil –
oil will spread and sample might be destroyed).
• 5 µL of sealing solution should be applied on top of the aqueous
phase with electronic multistep pipettes also.
• Please do always release the PCR mix or oil droplet first, at the
tip entry then deposit the droplet to the AmpliGrid reaction site.
For some pipette tips the oil droplet is not clearly visible. Even in
those cases move the pipette tip to the pre-deposited PCR mix.
The oil will move to the correct position. Ideal pipetting angle is
90°. If the pipetting angle is lower than that, droplet deposition
gets inaccurate.
Downstream analysis
PCR can be analysed by different techniques like:
• gel electrophoresis (agarose gel (~2%) for high expressed genes
or DNA amplification; polyacrylamide gels (8%) for lower
expressed genes (we recommend to do polyacrylamide gel
electrophoresis because of the superior detection limit). Pipette
4 µL of gel loading dye directly on the reaction site. Due to the
higher density of the buffer it will move through the oil and merge
with the sample. Use a standard 10µL pipette tip to aspirate the
5µL mixture of sample and buffer to load the gel. A minor transfer
of sealing solution does not interfere with the gel analysis.
• capillary electrophoresis; dilute the mix after PCR with 5 µL of
water by just pipetting on top of the sealing solution. The water
will merge with the PCR mix underneath the sealing solution.
Pipette off the mix and transfer it to the capillary electrophoresis
with an optional Sephadex column clean-up step. Please note,
that a longer sample uptake time might improve the quality of the
results.
• qPCR: after pre-amplification or reverse transcription on the
AmpliGrid the cDNA can be transferred to a real-time PCR
system by either recovering the 1µL from underneath the oil or
adding water to dilute the cDNA to a higher volume.
For research use only. Not for use in diagnostic procedures.
Beckman coulter biomedical gmbh • Sauerbruchstraße 50, 81377 Munich • Germany • Tel: +49 (0)89 579589-0, Fax: +49 (0)89 579589-3503 • E-Mail: [email protected] • www.advalytix.com
Worldwide Biomedical Research Division Offices:
Australia (61) 2 98446000 Canada (905) 819-1234 China (86) 10 6515 6028 Eastern Europe, Middle East, North Africa (41) 22 994 07 07 France 01 49 90 90 00 Hong Kong (852) 2814 7431/2814 0481 Italy 02-953921 Japan 03-5404-8359 Mexico (55) 560-57770 Netherlands 0297-230630 Singapore (65) 63393633 South-Africa, Sub-Saharan Africa (27) 11-805-2014/5 Spain 91 3836080 Sweden 08-564 85 900 Switzerland 0800 850 810 Taiwan (886) 2 2378 3456 Turkey 90 216 309 1900 U.K. 01494 441181 U.S.A 1-800-526-3821