### **Library Creation Process SOP:**

### **Qpix Colony Picker Protocol**

> Version Number: 1.0
>
> Start Production Date: x
>
> Author: Amy Geotina, Doug Smith
>
> Reviewed by: Nancy Hammon
>
> SOP Change Document: x

# Summary

This SOP describes the use of the Qpix colony picker as it applies to
the Library Creation group at the JGI.

# Materials & Reagents

> **[Materials/Reagents/Equipment]{.underline} [Vendor]{.underline}
> [Stock Number]{.underline}**

##### [Disposables]{.underline}

##### LB Amp 100 X-gal Plates (254mm) Teknova L133-A100X 

##### Nunc 384 Well Plate, Cell Culture Applied Scientific

##### [Stock Solutions]{.underline}

> LB Broth with 7.5% Glycerol Teknova 0181-G7.5

##### [Reagents]{.underline}

##### [Equipment]{.underline}

> Qpix

# Procedure

[Set up:]{.underline}

1.  To power on the Qpix turn the red lever on the right side of the
    picker from a horizontal to a vertical position. Press the red
    button marked reset on the front of the Qpix then the button on the
    lower right of the computer to power on the computer.

2.  Check that the air compressor on the left side of the Qpix is turned
    on and the release valve is shut. To turn on the air compressor
    twist the red button on the top of the pump counter clockwise.

3.  Launch the software program from the desktop icon Q-pix.

4.  To install the picking head click *diagnostics* from the toolbar at
    the bottom of the picking software's window. Move the actuator into
    an accessible position above by right clicking the mouse on a
    position above the picking bed and selecting *move actuator to* from
    the drop down menu that appears. Once the head is in the desired
    position open the door and insert the picking head into the actuator
    from the right side. Be sure and inspect the head first for any bent
    pins. Secure the head in the actuator by inserting the thumb screw
    into the hole on the left side of the actuator and tightening.

5.  Prepare the ethanol bath for picking by inserting the scrub brushes
    into the rectangular metal well and place the well into the front
    most well position on the right side of the picking bed. Fill the
    well with 70% ethanol so the level of the liquid just covers the top
    of the brushes. Check the ethanol level during use and add 100%
    ethanol if the liquid level drops below the brush tips.

> [Calibration:]{.underline}

1.  The Qpix does not require daily calibration as long as the plates
    being imaged and picked are checked for accurate picking and proper
    screening of the blue colonies. In general calibration once a week
    is sufficient.

2.  Refer to the Qpix manual located on the shelves behind the machine
    for calibration instructions.

3.  In general the size and shape parameters rarely need to be
    manipulated but the threshold values can change from plate to plate
    but tend to be consistent through an entire library.

4.  Tips for adjusting the threshold setting not mentioned in the
    manual: Make note of the different threshold values that are tried.
    There really seems to be no rule as how to best set the threshold
    values. The best settings are usually found by trying a few
    different values and selecting the value that gives you the highest
    number of picks. When comparing threshold values, be sure that the
    number of picks being compared is for an entire bioassay plate since
    the Qpix images different regions of the plate differently.

> [Run:]{.underline}

1.  Check agar volume is set to 300 ml. This can be done from the main
    menu.

2.  Set the *max. plates to use* to the desired number of output plates.
    The maximum that can be set up in one run is 15 destination plates.
    It is ok to set the plate number higher than the amount of colonies
    are available because the picker will indicate what plates it
    inoculated.

3.  Load the destination plate starting with the front right position
    and placing each additional plate in the column before moving onto
    next.

4.  Load plate with well A1 oriented towards the upper right corner. The
    plates should be snug in to corner. Negligence in this step will
    lead to picking head crashes and damage the head and needles.

5.  Check that the plexy glass is clean on the picking bed.

6.  Check that the bioassay plate is placed snuggly onto the bed. If the
    plate is loose it will likely shift during the run and become
    inaccurate.

7.  Once source and destination plates are properly placed in the
    picking bed select *full* from the bottom toolbar to image the
    plate*. Partial* may be selected to selectively image certain
    regions of the plate.

8.  Watch the computer monitor while the qpix is imaging to check each
    area of the bioassay is being processed correctly.

9.  If the imaging looked ok click *no* on the display image data
    prompt. Otherwise view the image again. If adjusting the imaging
    parameters refer to the manual located behind the qpix.

10. After a plate has been picked a dialogue box will alert you to the
    completion. From here, another box comes up asking whether to keep
    plate and position settings. Only if this was the last plate picked
    for a library should *No* be selected. Selecting *yes* will allow
    the picking for the next plate to be imaged to begin where picking
    just left off or for you to see what the last plate inoculated was
    and how many wells were inoculated.

> [Shutting down:]{.underline}

1.  Remove the sterilization bath and pour the remaining ethanol into
    the waste container. Remove the brush and rinse it with ethanol and
    remove any debris.

2.  Enter the diagnostic screen and position the actuator towards the
    center of the picking bed. Remove the picking head by loosening the
    screw to the left of the head and sliding the head out of the
    actuator while taking care to ensure that is does not drop. DO NOT
    place your hand under the needles. Place the head on the bed with
    the pins facing upward.

3.  Close the front door and click on UV light from the bottom of the
    toolbar. Allow the light to be on for 10 minutes.

4.  When the UV light is done select done in the bottom right of the
    Qpix software's window to exit the program. Once the program has
    been exited shut down the computer from the start button. After the
    computer has been given adequate time to shut down turn the red
    switch on the right hand side of the Qpix. Turn off power to the air
    compressor by pressing the red button on top then open the bleed
    valve to release any pressure.

# Reagent/Stock Preparation

How to prepare reagents and stocks