Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Chapter 8TroubleshootingImprecisePipettingThe calculated quantities of target nucleic acid are directly affected by how preciselythe template volumes are added to the reaction mixes. Other individually addedreagents are also affected by pipetting precision (such as, variable magnesium affectsamplification efficiency).Using Master MixesFor this reason, Applied Biosystems highly recommends using a master mix. Allcommon components to a set of reactions should be mixed together and thendispensed to the wells of the plate. Sub-master mixes can be used to further improvethe precision of identical replicates. For example, instead of pipetting 5 µL of thesame template into four replicate wells, pipette 20 µL of the template into asub-master mix, then divide the sub-master mix into four equal parts foramplification. When making each master mix, add 5–10% additional volume tocompensate for pipetting losses.Using PipettorsPipetting 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 methodConsult the manufacturer about the correct method of dispensing liquid volumesaccurately from the pipettor. For example, some pipettors are designed to deliver thedesignated volume at the first plunger stop, so ‘blowing out’ the residue may causeerror. Also, before using a new pipettor tip to serially dispense a master mix, wet thetip once by drawing up some of the master mix and dispensing it back into the mixagain.Non-OptimizedChemistryIncompleteMixingAir BubblesSplashing PCRReagentsChemistries that have not been optimized may be susceptible to inconsistencies. Tomaximize precision and reaction efficiency, optimize the primer and probeconcentrations of each individual assay used. Refer to the TaqMan Universal PCRMaster Mix Protocol (PN 4304449) for specific information about optimizing probeand primer concentrations for TaqMan-related chemistries.For maximum precision, the PCR master mix must be mixed to uniformity. After allreaction components are added to master mix, it should be vortexed for 4–5 secondsbefore aliquoting it to the wells of the plate. Any dilutions performed during theassay should also be vortexed.Air bubbles in the wells can refract and distort the fluorescent signals. Ideally, thereagents would be applied to the wells using a pipetting technique that does not formair bubbles. However, if a plate does contain air bubbles, they can usually be removedby swinging, tapping, or briefly centrifuging the reaction plate.If PCR reagents splash the undersides of the optical adhesive covers, the heat fromthe lid may bake the liquid to the cover and may distort the signal. If splashingoccurs, briefly centrifuge the reaction plate to remove all traces of liquid from thecaps.8-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm