Low Precision Or Irreproducibility - Applied Biosystems 7900HT User Manual

Drops
Writing on the
Reaction Plates
Fluorescent
Contamination on
the Plates
Errors
Contaminated
Sample Block
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Drops of reagents that cling to the sides of the wells may not contact the thermal
cycler sample block and consequently may not amplify. If the drop slides into the mix
during PCR, then the amplified products will become diluted and the result will be
less than replicate wells that did not have drops. Therefore, carefully monitor the
reaction plate as it is being transferred into the thermal cycler or 7900HT instrument.
If you observe any drops, take steps to remove them, such as centrifugation.
Do not write on any surface of the optical plates or the optical adhesive covers. The
fluorescent properties of the ink can potentially affect the fluorescence emission
from the plate and alter the results. Instead, note the contents of each well on a sheet
of paper, or on a printout of the sample setup.
Many compounds found in laboratories are fluorescent. If they come into contact
certain optical surfaces, such as the optical adhesive covers, the fluorescent results
may be affected. For example, it has been noted that the powder used to lubricate the
insides of plastic gloves often contains fluorescent compounds. Use only powder-free
gloves and do not needlessly touch the reaction plates or optical adhesive seals.
Human errors from time to time are inevitable, such as pipetting into the wrong well,
or making a dilution mistake.
Human error can be reduced in the following ways:
• Perform the assay in a systematic fashion. For example, the pattern of sample
positions should be simple (avoid putting gaps in the rows).
• When pipetting the master mix, look directly down into the reaction plate so that
you can verify the transfer of the solution.
• If adding a small-volume reagent, such as template, place the drop of liquid on
the side of the well. Briefly tap or centrifuge the plate afterwards to bring the
droplet down into the well.
• After all pipetting is complete, visually inspect all the wells to confirm the
presence of the reagent drops. Tapping or centrifuging the reaction plate will
cause all the drops to slide down into the wells simultaneously.
• When making serial dilutions, be sure to change the pipet tip after each dilution
step.
• Visually inspect the liquid volumes being pipetted to verify that the volume is
approximately correct. A common mistake is using the wrong pipettor volume
setting (such as setting 20 µL instead of 2.0 µL).
• Visually inspect the volumes of the completed reactions, looking for any wells
that have volumes that do not match those of the other wells.
Any material contaminating the sample block can affect the results. For example,
mineral oil reduces thermal transfer. Residue from writing on reaction plates darkens
the wells, absorbing light.
The sample blocks should be periodically inspected for cleanliness. Sample block
contamination can be visualized by running a background plate and inspecting the
resulting background signal for aberrant peaks above 2500 FSU (see
page 7-14 for instructions on decontaminating the sample block.
DRAFT
September 1, 2004 11:39 am, CH_Trouble.fm

Low Precision or Irreproducibility

page 7-16). See
8-7