| Inoculations of Bioblocks |
| Version Number: | 2 |
| Start Production Date: | X |
| Author: | Matt Fourcade |
| Edited by: | X |
| Reviewed by: | X |
| Summary |
| Materials & Reagents |
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Reagents | ||
| Terrific Broth, augmented with 1.6% Glycerol | Teknova | N/A |
| 10X Phosphate buffer | Teknova | N/A |
| 50mg/ml Ampicillin | Sigma | N/A |
| Equipment | ||
| PrepTrack robot | JGI | N/A |
| 96 well microtiter plates | Falcon | N/A |
| Multidrop robot | Titertek | N/A |
| Procedure |
Setup:
1. Using a Multidrop dispensing robot, fill 96-well microtiter plates with 185ul per well of Terrific Broth (augmented with 1.6% Glycerol, 10X Phosphate buffer and 50mg/ml Ampicillin). If needed, filled plates can be covered and stored at 4 degrees C for up to 3 days.
2. Remove glycerol stock plates to be used from the -80C freezer. Thaw on benchtop.
3. Obtain an equal number of previously filled 96-well microtiter plates.
4. Label the microtiter plates with the name and number of the plate to be inoculated using computer printed labels.
Inoculation:
Run PrepTrack robot to inoculate blocks
Incubation:
1. Place each inoculated plate, without lids, into the acrylic Hi-Gro plate carrier. (Leave the top plate nest in the plate rack empty to avoid contamination via condensation of a plate in that position.)
2. Place the acrylic rack(s), containing plates, into the Hi-Gro incubation cylinder(s). Be sure that the front of the rack is parallel to the plate holders at the front interior of each Hi-Gro cylinder.
3. Secure the plate holders in place by turning the thumb screws (located on the front exterior of each Hi-Gro cylinder) clockwise until both the rack and the plates it contains are immobile (verify this both manually and visually).
4. Place a cylinder lid on each Hi-Gro cylinder and secure the lid with metal fasteners.
5. To turn Hi-Gro on, make sure that the speed control is first set to "0", then Press the "Power" button.
6. Slowly turn the speed control knob clockwise until a shaking speed of "6.0" is built up.
7. Make sure that the speed control is set @ 37 degrees C.
8. Turn on the oxygen valve 1 hour after the Hi-Gro’s have begun to incubate.
9. Incubate the plates for a total of 18 hours.
Clean-up and Storage
1. Place foil sealers on each of the glycerol stock plates and place them in a freezer rack.
2. Catalog the location of each plate (including freezer rack number and location within the freezer rack) using the barcode on each plate to add to the freezer catalog database on the computer
3. Store the glycerol stocks at -80 degrees C.