16 April 20012 April 2001

This protocol describes how colonies are to be picked by hand into 384-well plates with toothpicks.

1. Obtain NZY-Amp agar plates prepared as described in the protocol for Mass Excision and Transfection of Unamplified EST Libraries, optimally with 300-350 colonies per plate.
2. Prepare 384-well nunc plates:

• Label the left and front of the plate with a paint pen, include the library designation (e.g., FM1 – do not forget the numeral!), date (Month.Day.Year), the initials of the person doing the picking, and the plate designation (AA, AB, etc.). This designation can be added after overnight growth if it is not known in advance.
• Fill each well with 100ul of freezing medium (see recipe protocol).

1. Use sterile (autoclaved) toothpicks, one per colony to pick. Ensure that bacteria from only a single, isolated colony are picked and that the tip of the toothpick is well coated with bacteria from that colony. Do not pick colonies too close together. Do not pick blue colonies. Do not pick colonies that are not uniformly round as they might have originated from two different bacteria. Do obtain instruction from someone who already does it very well.

• Note contaminated wells and any questionable wells with reference to the plate designation on the 384-well picking worksheet.

1. If you pick at a good rate, it usually works well to inoculate two rows at first with toothpicks, then remove toothpicks from the first column and do the third. [If you are more comfortable with a different strategy that’s OK. But ensure that you keep precise track of where you are by beginning the next row before removing all toothpicks from the plate.] Continue by removing toothpicks from one column and inoculating the next one, one column at a time. When you remove the toothpicks it is essential that you be careful not to cross contaminate wells. If you think you might have done so, please record this information with reference to the plate designation on the 384-well picking worksheet.

• How long the toothpicks stay in the freezing medium is critical, too long and the media is absorbed by the toothpick, not long enough, and the well is not inoculated properly.

1. When all wells have been inoculated, cover with a gas permeable seal. Insert the plate into the HiGro cassette holder. Store at 4°C until time to incubate.
2. Place the cassette holder with the inoculated plates into the HiGro incubator, and culture overnight (37°C, 5.2 RPM, 20 hr incubation). Handle the 384-well plates with great care, especially when putting into or taking out of the HiGro cassette. Any sharp bump will cause medium with cells to bounce up onto the filter.

• Upon removing the plate the following morning, each plate should be examined carefully for growth quality and any wells with no growth, poor growth or any questionable wells should be noted with reference to the plate designation on the 384-well picking worksheet. (Refer to the legend on the form.)

1. For wells that had no growth, aspirate and discard the medium using a pipet with a yellow tip. Mark these same wells on the bottom of the plate with a black pen so that they can easily be identified later.
2. The forms are accumulated in a separate binder for each library.
3. Once the results have been entered on the forms, remove gas permeable and replace with Robbins foil seals.
4. Place the plate’s right side up in the –80°C freezer.

384-well picking worksheet