Molecular Devices ClonePix 2 Specification sheet

07MAN1181.A1
Effective Date: 30-Jan-11
ECO #: 3093
ClonePix
TM
2
QUICK SET-UP INTRUCTIONS MANUAL
SOFTWARE RELEASE 1.2.80.1071

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Contents
What are Q ick Set-Up Instr ctions? .................................................. 3
Starting p ClonePix 2 ........................................................................ 3
Loading Plates .................................................................................... 4
Preparing for a Pick R n ..................................................................... 5
The Pick R n ....................................................................................... 6
Imaging Settings .................................................................................................. 7
Picking Settings .................................................................................................... 8
Sanitize Pin Options .............................................................................................. 9
Start Pick R n ....................................................................................................... 9
Preview ........................................................................................................ 9
Sele t Wells ................................................................................................. 11
Summary .................................................................................................... 11
Imaging....................................................................................................... 11
Results ........................................................................................................ 12
Pi king Review ............................................................................................. 14
Pi king ........................................................................................................ 14
Finish .......................................................................................................... 14
Powering Down ClonePix 2 ............................................................... 15
Contact Details ................................................................................. 16

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What are Q ick Set-Up
Instr ctions?
These instru tions are designed to enable a new user to undertake a simple pi king run on
ClonePix 2. The following instru tions offer guidelines only. No attempt should be made to use
ClonePix 2 before the system has been fully installed by an Approved Engineer.
For full information please read the ClonePix 2 Robot Man al and Software Applications
Man al. Please refer to the website for the latest reagents & supplies, repla ement parts and
optional extras.
Starting p ClonePix 2
 Ensure robot and ompressor are plugged in.
 Turn on the ompressor and ensure ompressed air gauge is set to 80 psi (5.5 bar).
 Ensure Emergen y Stop button is not pressed in.
 Swit h on ClonePix 2. The HEPA filtration system works all the time that ClonePix 2 is on.
 After approximately 2 minutes, initiate ClonePix 2 software.
 Wipe out ClonePix 2 bed with 70% ethanol or fresh Sterilizing Agent (K8080) using a lint-
free loth.
 Fill the ethanol feed bottle with 70% ethanol and empty the ethanol waste bottle.
 Make sure that the orre t Pi king Pins are installed for the type of ells to be pi ked.
F1 Picking Pins (400µm internal diameter; X4961) for suspension ell pi king from
semi-solid medium.
F2 Picking Pins (700µm internal diameter; X4962) for adherent ell pi king from liquid
medium.
Pi king Pins should be leaned by soni ation in aQuClean (K2505) and auto laved. It is
advisable to auto lave the Pi king Pin Removal Key (X4948) at the same time. See General
Maintenan e se tion of the Robot Manual for instru tions.
If the Pi king Pins need to be hanged remove the Pi king Head and swap the pins. To do
this, li k on the Pi king Head Management i on, then the Repla e Head i on and follow the
on-s reen instru tions. Refer to the General Maintenan e se tion of the Robot Manual for
guidan e on how to remove and repla e the head and the pins.

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Loading Plates
It is important to know how to load plates into ClonePix 2 orre tly.
A ClonePix 2 bed layout is as shown below:
The Sour e plate sta ker system is lo ated at the rear of the ma hine, and the Destination
plate sta ker system is at the front. Both the Sour e and Destination sta kers have 1
removable assette (left hand side).
Ea h assette an a ommodate up to 10 mi roplates. The plates are fed individually from
the assette, via the plate holder, onto the bed with automati lid removal as the plate passes
the plate lid lifter. On ompletion of olony pi king, the de-lidded plate is then returned to the
assette where the lid is repla ed.
Important notes:
 Plates loaded into a assette, via the plate holders, must be of the same type and must
mat h the plate type sele ted in the software. Loading of a plate type different from that
sele ted in the software is likely to damage the plate and the instrument.
 Plate holders must be pla ed level into the assette and all the way to the ba k of the
assette to ensure orre t positioning.
 When the assettes are pla ed in the sour e and destination sta kers, they must be held
firmly in pla e by the lo king bolt on the front left hand side of the sta ker systems.
Failure to lo k a assette may ause a malfun tion of the olle tion and/or return of the
mi roplates.
 Sour e and destination plates must be loaded onto plate holders and then into the
assettes with lids on and well A1 in the front right-hand orner as shown below:
Imaging station
Wash station
Sour e Cassette
Destination Cassette

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Orientation of sour e plates in ClonePix 2
Orientation of destination plates in ClonePix 2
Preparing for a Pick R n
Prior to arrying out a pi king run, it is important to make sure that ClonePix 2 is set up
orre tly. The Prepare for Pick R n pro ess is designed for this purpose. This pro ess helps
the user to validate that 1) the pins are firing orre tly, 2) the amera, pins and mi roplates
are aligned, and 3) the fluid system is sterile and ready for use.
 Cli k on the Prepare for Pick R n i on and follow the on-s reen instru tions.
When asked to load a sour e plate, it is re ommended that a blank PetriWell-6 plate is used.
However, the use of any onfigured plate (those present in the drop-down list) should work.
On e aligned using a onfigured plate type, ClonePix 2 is then ready for use with any
onfigured plate type.
The Prepare for Pick R n pro ess does not need to be arried out prior to every pi king
run. It must be arried out every time that ClonePix 2 is first powered up.
 Cli k Close Pro ess to return to the Main Navigation S reen.

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The Pick R n
The Pick R n pro ess is designed to aid new users through their first pi king run. Use of the
default parameters provided should fa ilitate su essful imaging, olony sele tion and pi king.
The des ription below assumes that you have plates of ell olonies with fluores ent halos of
se reted protein.
 Cli k on the Pick R n i on to open the following s reen:
The settings an be edited by li king on e on the se tion headings.

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Imaging Settings
 Cli k on the Imaging Settings title to open the following s reen:
Fill in the following information:
 R n Annotation
Enter a name to identify this run.
 So rce Microplates
Enter the sour e plate type that will be
used for this run.
 Barcode Options
Sele t Read Barcode.
Sele t A to-assign barcode in case of
fail re.
If there are no bar odes on the plates,
ClonePix 2 will automati ally assign a
ode.
 Batch plates
Sele t this option. This assumes that
multiple plates ontaining the same
sample are being loaded and they will be
pro essed as a single experiment.
 So rce Plate Options
Sele t Finish when assette is empty.
 Acq isition Options
This option provides a hoi e of whi h images to apture. If there are appropriate Image
A quisition options sele t them here. If not, the a quisition options an be reated later
in Preview (see below). Most ommon options sele ted are one white light and one
fluores ent option.
 Prime Config ration
The Prime Configuration is the a quisition option to be used for olony dete tion. Sele t
the white light option if available.
 Review Colony Selection
Sele t Bat h – Review All.
 Cli k Apply.

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Picking Settings
 Cli k on the Picking Settings title to open the following s reen:
Fill in the following information:
 Destination Microplates
Enter the plate type that will be used for this
run.
 Destination Wells
Spe ify the wells into whi h olonies are going
to be deposited. Right li k on the mouse to
sele t wells, and left li k to de-sele t wells.
All destination plates will be filled using this
template.
 Barcode Options
Sele t Read Barcode.
Sele t A to-assign barcode in case of
fail re.
If there are no bar odes on the plates,
ClonePix 2 will automati ally assign a ode.
 Deposit Options
Leave unsele ted.
 Destination Plate Options
Sele t Finish when assette is empty.
 Pick N mber Options
Do not sele t any options here. Leave as
default (Collate by Plate).
 Pin Options
Sele t the type of olonies that are going to be pi ked: Suspension or Adherent.
Pi k Height Adjustment: Leave as default.
Aspirate Volume: Leave as default = 5µl.
Dispense Volume: Leave as default = 7µl.
 A dit Options
Ti k this box to save the Target and Aspirate Images. Note: This will slow down pi king.
 Dispersal Options
Use dispersal: Sele t to spread out olony ells after pi king. Do not sele t if inta t
olonies are required.
Dispersal y les: Use 3-6 for CHO ells and 6-10 for hybridomas.
Dispersal volume: Leave as default = 20µl.
 Cli k Apply.

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Sanitize Pin Options
Leave as default – no hanges required.
Start Pick R n
Cli k Start to begin the pro ess.
When prompted, load the ell olony plates into the sour e sta ker assette.
Preview
This is where image a quisition and olony dete tion settings are set up. The preview s reen
will be displayed like this:

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Step 1: Set up Image A quisition Settings
 Cli k on the A quisition tab.
 If the desired a quisition option is not available in
the My Configurations box, new A quisitions options
will need to be set up (see example s reenshot on
right).
 Cli k New and then li k Edit. The following settings
should work for most s enarios:
Des ription White Light FITC 1s
Ex itation Filter
WHITELIGHT
(TRANS)
EGFP/FITC
Exp. Time (ms)
200 1000
LED Intensity 3 128
Camera Fo us 2200 2200
 Cli k Save to store ea h option.
 The new a quisition options will now be displayed
under My Config rations. Ensure that TransWL and
FITC 1000ms options are sele ted.
 Note: Do not delete the Default option – it is required for other ClonePix 2 fun tions.
 Set the Prime Config ration to TransWL. This is riti al for orre t olony dete tion.
 To apture the images either li k Grab Image whi h will apture images for the area
urrently highlighted in the Well Map, or li k on another area on the Well Map to display
a new image.
 There will now be a main image with 3 thumbnails to the left
named Composite, TransWL and FITC 1000ms. Toggle between
the thumbnails to inspe t the images.
 Red pixels on the image indi ate that it is overexposed. In this
ase, lower either the exposure time or LED Intensity and Grab
Image again. Adjust until there are no red pixels.

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Step 2: Set up Dete tion Settings
 Cli k on the Detection tab.
 Set Algorithm to Local Threshold from the drop-down menu.
 Using the White Light image, set the Average Colony Diameter to a size that best
dete ts the olonies. This will probably be between 0.25 and 0.70mm depending on the
size of the olonies. Cli k Reprocess after moving the slide bar to apply the hanges.
 Leave Exterior Statisti s Diameter Multiplier at the default setting (x3).
 Leave Use ea h olony size when al ulating exterior statisti s desele ted.
 Leave Display settings as default (only Display Dete ted Colonies and Shade Overlap
Areas sele ted).
 Cli k Next to pro eed.
Select Wells
Sele ted wells are shown as red.
 Cli k Next to pro eed.
S mmary
 Cli k Next to pro eed.
Imaging
ClonePix 2 will now automati ally image the sour e plates, dete t the olonies and generate
ombined data for all plates.
 While ClonePix 2 is imaging, pre-fill one or more 96-well destination plates with liquid
medium (150µl re ommended) and pla e in an in ubator to equilibrate.

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Res lts
Imaging results are displayed in the Res lts s reen:
 Cli k on the Gro ps tab in the Graphs tab.
 Any group an be altered but this may ompromise lonality and viability. Where possible
it may be best to leave the groups with the default values.
 Groups an be edited by double li king on the desired group and moving the default
gate.
 For Example: the Too Small group ut-off point an be redu ed by double li king on the
group (whi h will display the urrent ut-off value) and then dragging the slide bar on the
histogram to 0.05 or the desired value.

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 Colonies in the Ungated group are those that have good size, shape and distan e from
other olonies.
 To further isolate only the olonies in the Ungated group that have highest asso iated
FITC fluores en e, li k All Undiscarded Feat res to display all the olonies on the
histogram.
 Sele t [FITC] Exterior Mean Intensity from the Histogram drop-down menu. Using
the Add new line gate tool, draw a gate on the histogram by li king on the histogram
where the ut-off point will be and then drag the gate off the right end of the histogram –
Cli k and Drag.
 An automati window will appear where it
is possible to name the group by typing
‘High FITC’ in the box and give it a
different olor. Cli k OK.
 Cli k on the newly- reated group to sele t
it and li k Decrease Priority multiple
times so that it sits just above the
Ungated group.
 Cli k Next to pro eed.
Click & Drag

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Picking Review
 In the Picking Review tab, sele t only the High FITC group:
 Under Sort Options, sele t Order By [FITC 1s] Exterior Mean Intensity.
 Cli k Next to pro eed.
Picking
 When prompted, load the destination plate(s) into the destination sta ker assette (the
number of destination plates required will be displayed in the Pick S mmary on the
Picking Review tab).
 Cli k Next to pro eed.
 The pi king step will pro eed automati ally until all olonies in the sele ted group have
been olle ted.
Finish
 Cli k Finish to return to the Pick R n pro ess top page.
 Cli k Close Process to return to the Main Navigation Screen. If settings have not
been saved previously a prompt will warn of this and allow settings to be saved.
 To view the results of the pi king run, li k on the Review Res lts i on.

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Powering Down ClonePix 2
 Exit from the ClonePix 2 appli ation by sele ting Exit from the File menu on the main
setup s reen.
 Close down Windows – Cli k the Start menu at the bottom of the s reen then li k Sh t
Down. Wait for omputer to swit h off ompletely.
 Turn the instrument off by pressing the Stop button on the front of the system.
 Turn the power off at the mains.

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Contact Details
Molec lar Devices
Queensway, New Milton
Hampshire BH25 5NN, UK
Tel: [phone]
Fax: [phone]
Web: www.mole ulardevi es. om/genetix
For all te hni al queries please onta t your nearest Customer Support group. Visit
www.mole ulardevi es. om/genetix for latest onta t details.
Trademarks
ClonePix™, CloneSele t™, CellReporter™, HalfBD®, 'NRi h®, SlidePath™, Data Arena™ and Image Arena™ are trademarks of
Mole ular Devi es (New Milton) Ltd.
Copyright © 2011 by Mole ular Devi es (New Milton) Ltd
All rights reserved. No part of this publi ation may be reprodu ed, stored in a retrieval system, or transmitted,
in any form by any means, ele troni , me hani al, by photo opying, re ording, or otherwise, without the prior
written permission of Mole ular Devi es (New Milton) Ltd.
Information furnished by Mole ular Devi es (New Milton) Ltd is believed to be a urate and reliable; however, no responsibility is
assumed by Mole ular Devi es (New Milton) Ltd, for its use; nor for any infringements of patents or other rights of third parties whi h
may result from its use. No li ense is granted by impli ation or otherwise under any patent rights of Mole ular Devi es (New Milton)
Ltd.
Revised June 2011