Overview

The Genomics Core Facility (GCF) is equipped with two liquid handling robots, a MultiProbe II and a Biomek FX. The robots are operated by GCF personnel on a fee for service basis.

The Perkin Elmer MultiProbe II EX is an eight-tip instrument designed to pipet 2ul up to 250ul volumes into 96 and 384 well plates, Eppendorf tubes, and other labware. This robot is used routinely to pipet 384 well RT-PCR plates with a 10ul total reaction volume. When the cost of supplies, reagents, and technician time is calculated, robotic pipetting is the most accurate and cost effective answer for the pipetting of 384 well RT-PCR plates. GCF will schedule up to six 384 well plates for robotic pipetting in one day or the equivalent of twenty-four 96 well plates enabling experiment data to be available to the researcher rapidly. The MultiProbe’s configuration gives a high degree of programming flexibility. Custom pipetting programs can be written for special projects. Please contact GCF personnel for project consultation well in advance of your planned start date.

The Beckman Coulter Biomek FX is available for custom programming of high through-put pipetting tasks. It is configured with a 96 tip head which will make pipetting transfers and liquid additions to and from 96 and 384 well formatted plates. It is equipped with a vacuum station which may be used for protocols requiring liquid drawn through a column as commonly found in many purification protocols.

Scheduling

For pipetting 384 well RT-PCR plates, Pennington employees may schedule an appointment for the instrument (R_GCF_Rosie) on the Outlook calendar at least 24 hours in advance. Allow one hour for each plate you wish to pipet.

Detailed instructions for using the Outlook calendar to schedule an instrument (resource) may be found in the Scheduling Resources - Frequently Asked Questions.

For scheduling less than 24 hours in advance or for customers outside of Pennington please contact GCF for an appointment.

Cost

Requirements for Robotic Pipetting of 384 Well RT-PCR Plates

The MultiProbe II robot is programmed to pipet 10ul reactions into an Applied Biosystems 384 well Optical plate. Save the spreadsheet Robotic RT-PCR Calculator.xls to your computer to calculate the correct amount of template and master mix for your experiment. It is extremely important that you use properly calibrated pipettors for RT-PCR experiments. The spreadsheet provided calculates the correct amount of reagents needed, but if your pipettors are off, you may find that you haven’t provided the proper amounts of each reagent. QPCR is quantitative. Your data accuracy is highly dependent upon the accuracy of your tools.

The following must be supplied to the Genomics Core Facility:

• RNA or cDNA template
  1. The template should be provided in an Applied Biosystems 96 well optical plate. The total amount of template added is proportionally scaled down from the amount used in your manual reactions. If you normally use 30ul total reaction volume, you are scaling down to 10ul; therefore, divide the quantity of template by 3.
  2. The robot pipets 3ul of your template into each reaction. You will need to adjust your template concentration to ensure that the amount of template you wish to put into your reaction is contained in 3ul. (For example, in a manually set up reaction you normally use 2ul of a 2ng/ul concentration template for a total of 4ng template in a 10ul reaction. The new concentration needed for the robot is 1.33ng/ul giving the same 4ng total in your robotically pipetted reaction mix.) You may make template master plates that can be used to set up multiple 384 well plates on the robot. The plates are sealed and stored at –70C between runs.
  3. The spreadsheet includes 10ul excess template in each well to insure that the robot pipet tip is able to pick up template for all wells in your experiment.
  4. For duplicate reactions on the 384 well plate, your templates should be placed in column order on a half-skirted 96 well plate (A1, B1, C1….H1). Do not skip any wells. Place your standards and NTC last.

     For triplicate reactions on the 384 well plate, your templates should be placed in double columns as shown below (A1, A2, B1, B2, …., H1, H2, A3, A4, …..H3, H4….). Do not skip any wells. Place your standards and NTC last.

     
• Master Mix
  The master mix contains all of your reagents except the template. The robot will pipet 7ul per well into the 384 well plate. Place each master mix into eight vertical wells (a column) of a 96well plate. The spreadsheet (Robotic RT-PCR Calculator) will indicate the correct volume for each well for duplicate or triplicate reactions.

  

  OR

  

  OR for large amounts of Master Mix

  

  Divide the master mix volume for each well as determined on the Robotic RT-PCR Calculator spreadsheet equally between two adjacent horizontal wells.

• 384 Well Plate Layout

  The robot pipets into the plate in column order. Your 384 well plate will be laid out in that order (A1, B1, C1,…., P1). For duplicate reactions the robot will pipet from well A1 in your template plate to wells A1 and B1 in your 384 well reaction plate.

  

  Triplicate reactions will be placed in A1, A2, A3, B1, B2, B3, C1, C2, C3…..on your 384 well plate.

  

• Supplies
  The Genomics Core Facility will provide robotic pipet tips, 384 well PCR plates, and plate sealing tape.
  
  
• Submission to the Genomics Core Facility
  The following must be provided to the Core Laboratory 24 hours prior to your scheduled run on the 7900:
  • Total number of samples (include standards and no template control)
  • Number of genes to be tested on the plate
  • Number of replicates
  • Column location of each master mix in a 96 well plate. Please indicate whether the master mix is included in your template plate or in a separate plate.

  Alternatively, you may provide an Excel plate map of each plate containing samples and master mixes.

• You must be present at the end of the robotic pipetting to remove your plate, check it for pipetting errors, seal it, centrifuge it, and start your run on the 7900.
  
  
• When 384 well plates are pipetted by robot, the samples are in vertical well order. To ease data entry for these plates refer to the document Using Excel to Set Up a Real-Time PCR Plate

Links

• Master Mix and Template Volume Calculator for Robotic Pipetting