ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Writing on the
Reaction Plates
Fluorescent
Contamination on
the Plates
8-6 Troubleshooting
Drops 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 final 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 384/96-Well Reaction 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 in contact with
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.
Errors 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 (such as 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.