Applied Biosystems 7900HT User Manual page 294

Chapter 8 Troubleshooting
Imprecise
Pipetting
Non-Optimized
Chemistry
Incomplete
Mixing
Air Bubbles
Splashing PCR
Reagents
8-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, CH_Trouble.fm
The calculated quantities of target nucleic acid are directly affected by how precisely
the template volumes are added to the reaction mixes. Other individually added
reagents are also affected by pipetting precision (such as, variable magnesium affects
amplification efficiency).
Using Master Mixes
For this reason, Applied Biosystems highly recommends using a master mix. All
common components to a set of reactions should be mixed together and then
dispensed to the wells of the plate. Sub-master mixes can be used to further improve
the precision of identical replicates. For example, instead of pipetting 5 µL of the
same template into four replicate wells, pipette 20 µL of the template into a
sub-master mix, then divide the sub-master mix into four equal parts for
amplification. When making each master mix, add 5–10% additional volume to
compensate for pipetting losses.
Using Pipettors
Pipetting precision is also improved by:
• Calibrating and servicing the pipettors regularly
• Pipetting larger volumes
• Reducing the number of pipetting steps whenever possible
• Increasing the consistency of the pipetting method
Consult the manufacturer about the correct method of dispensing liquid volumes
accurately from the pipettor. For example, some pipettors are designed to deliver the
designated volume at the first plunger stop, so 'blowing out' the residue may cause
error. Also, before using a new pipettor tip to serially dispense a master mix, wet the
tip once by drawing up some of the master mix and dispensing it back into the mix
again.
Chemistries that have not been optimized may be susceptible to inconsistencies. To
maximize precision and reaction efficiency, optimize the primer and probe
concentrations of each individual assay used. Refer to the TaqMan Universal PCR
Master Mix Protocol (PN 4304449) for specific information about optimizing probe
and primer concentrations for TaqMan-related chemistries.
For maximum precision, the PCR master mix must be mixed to uniformity. After all
reaction components are added to master mix, it should be vortexed for 4–5 seconds
before aliquoting it to the wells of the plate. Any dilutions performed during the
assay should also be vortexed.
Air bubbles in the wells can refract and distort the fluorescent signals. Ideally, the
reagents would be applied to the wells using a pipetting technique that does not form
air bubbles. However, if a plate does contain air bubbles, they can usually be removed
by swinging, tapping, or briefly centrifuging the reaction plate.
If PCR reagents splash the undersides of the optical adhesive covers, the heat from
the lid may bake the liquid to the cover and may distort the signal. If splashing
occurs, briefly centrifuge the reaction plate to remove all traces of liquid from the
caps.
DRAFT