DiDonato, Ray 1-2
Growing Plates for the Robot
(96-well plate plasmid preps for sequencing)
1. Prepare the 2X YT Plus Media as described below. Store at 4oC.
2. When you need to pick any plates for the robot add the appropriate antibiotic to the
sterile 2X YT Plus Media. Use the same antibiotic concentration that was used for
your cloning plates. You will need approximately 100mL of media for each plate.
(i.e. Add 100uL of 50mg/mL Ampicillin to 100mL media for one plate)
3. Using a Bunsen burner to create a sterile environment and wearing gloves. Aliquot
1mL of 2X YT Plus media with antibiotic to each well of a pre-sterilized 96 deep well
plate for the robot. (These pre-sterilized plates can be found room 412)
4. Carefully pick colonies from the cloning plates into individual wells using sterile
200uL tips or toothpicks.
Note: Below are some ideas for the number of colonies you need to pick to test
different clones. (Be sure to pick the clones in groups by columns.)
a. To check if a primer set amplifies the correct gene, pick 12 wells (1A-2D).
b. To check for culture purity with a 16S library, pick 24 wells (1A-3H).
c. To check the community of a culture with a 16S library, pick 48 wells (1A6H).
5. Carefully remove the tips used to pick the colonies from the wells and cover the plate
was an Airpore Tape sheet. The back of this sheet can be removed so that the sheet
can be placed directly over the wells. Please leave the small piece of backing on the
tape so that it can be easily removed later.
6. If the plate is picked early in the day it needs to be stored at 4oC until it can be move
to the 37oC incubator. To prevent the cells from experiencing a large shock that can
interfere with growth, be sure to remove the plates from the 4oC and warm to room
temperature for 30 to 60 minutes.
Note: For clones with larger inserts that are greater then 1.5kb prewarm the 96-well
plate with media and colonies to room temperature for 30 to 60 minutes before
picking the colonies. If you still have difficulty with the plates growing well, then
you can prewarm the 96-well plate with media in the 37oC incubator for 30 to 60
minutes before picking the colonies. Also after picking these plates it is best to put
them immediately into the 37oC incubator or keep them at room temperature.
7. Grow the plate overnight at 37oC on the rotating shakers. If the plate is shaking at
about 200rpms, then the cells should grow in about 14-16 hours. The incubation time
and shaking speed can be adjusted if needed. The faster the cells are shaking the
faster they will grow, but the shaking should not be faster then 250rpm. Above this
speed the cell growth is diminished.
8. Remove the plates from the 37oC and keep at 4oC. Check a few wells of the plate to
see of the cells have grown sufficiently by measuring the absorbance at 600nm on a
spectrophotometer. (Be sure to use the 2X YT Plus Media as a blank.)
The 600nm absorbance should be around 1.5 to 3.5 for the robot to make a good
plasmid preparation.
9. Return the plates to storage at 4oC until they can be run on the robot.
DiDonato, Ray 2-2
They should not be kept for more then 3 days before being processed by the robot. If
you need to keep the plates longer you will need to spin them down for 20mins at
3200rpm, pour off all the supernatant you can, and freeze them at –20oC.
2X YT Plus Media (1L)
31g
2X YT
5g
Na2HPO4
10g
KNO3
8g
Glycerol
upto 1L
dH2O
Sterilize the media by autoclaving for 30 minutes on a liquid cycle.
Allow the media to cool and then store at 4oC.
50mg/mL Ampicillin (15mL)
750mg
Ampicillin
upto 15mL
dH2O
Sterilize the solution by filtering, then split the solution into 1mL aliquots and store
these at –20oC.