EST Sequencing: Picking Protocol

General Description: The plasmid containing the insert DNA has been incorporated into DH10B(tm) E. coli host cells and streaked onto agar plates with 100 microg/ml (final concentration) ampicillin for incubation. The resulting colonies are then picked into 96-well shallow plates for further incubation and transferred to QIAGEN(R) blocks for QIAGEN(R) prepping. This protocol describes the procedures for media preparation, picking into shallow well plates (with incubation) and stamping cells into 96-Deep-well blocks (with incubation). Note: In order to ensure accurate identification of plates during genomic sequencing, barcode labeling is utilized throughout this protocol.

Materials & Reagents:

Material / Reagent	Vendor	Stock #
Terrific Broth w/salts (TBS) media	Prepared at Genome Center	N/A
100% glycerol stock	Roche	100 649
Sterile wooden toothpicks	Prepared at Genome Center	N/A
Ampicillin (sodium salts) (25 mg/ml)	Sigma-Aldrich	A9518
96-well plate	Costar	3794
Incubated colonies	See Colony Growth protocol	N/A
Sterile reservoirs	Matrix	8096
Deionized and distilled water (ddH2O)	Prepared at Genome Center	N/A
Bacto™ Yeast Extract	Fisher Scientific Co.	DF0127-17-9
Bacto™ Tryptone	Fisher Scientific Co.	DF0123-17-3
96-deep-well blocks	QIAGENâ	19573
Plate sealers	Edge Biosystems, Inc.	48461
96-Well disposable pin replicators	Incyte Genomics	ATD-5000

Procedure

Preparation of TBS media

1. Add 50 ml TB salts (see below) to 450 ml TB (see below) and mix thoroughly.
2. Add 1 ml (25 mg/ml) ampicillin to each 500 ml volume of TBS media solution. (Note: Final concentration of ampicillin in TBS media should be 50 micrograms/ml.)

Preparation of Terrific Broth (TB)
Prepared and supplied at the Stanford Genome Technology Center

1. Add 6 g Bacto. Tryptone, 12 g Bacto. Yeast extract and 2 ml glycerol to 450 ml ddH2O.
2. Mix thoroughly and autoclave.

Preparation of TB Salts
Prepared and supplied at the Stanford Genome Technology Center

1. Add 1.2 g Potassium phosphate (Monobasic) and 8.2 g Potassium phosphate (Dibasic) to 50 ml ddH2O.
2. Mix thoroughly and autoclave.

Picking Colonies into Shallow 96-well Plates and Incubation

1. Add 48 ml glycerol to TBS (2 ml of glycerol already added during preparation of TB) to give a final concentration in solution of 10 % glycerol.
2. Dispense 200 microliters of 10 % Glycerol-TBS solution into 96-well plates.
3. Retrieve colonies from incubator.
4. Label each 96-well plate with library number for those plated colonies.
5. Using a different, sterile toothpick for each colony, inoculate each well in the 96-well plates with a separate colony.
6. After the entire plate has been inoculated, remove toothpicks.
7. Place 96-well plates in incubator at 37 C for 20 hours.
8. After incubation, plates may be stored at .80 C for long term storage or proceed to deep-well block stamping.

Stamping into Deep-well QIAGEN(R) Blocks and Incubation with Shaking

1. Retrieve relevant plates from .80 C freezer or incubator.
2. For frozen plates, peel off freezer tape and let thaw for approximately 25 minutes at room temperature. Keep a paper towel over plates to soak up the condensation. Put clones in incubator for 30 minutes to revive.
3. Fill and dispense 1200 microliters of TBS (see above) into square-well blocks.
4. Using a separate 96-well pin replicator for each library plate, orient according to rows and columns and gently place in retrieved shallow well library plate.
5. Agitate pin replicator gently and let sit a few seconds.
6. Carefully remove pin replicator and transfer to labeled 96-square-well block with TBS.
7. Cover blocks with AirPore(tm) tape and shake for ~20 hours at 37 C, 300 rpm. (Note: Blocks may also sit at room temperature for a few hours before shaking).
8. After shaking is complete, spin blocks in Jouan centrifuge for 7 minutes at 2700 rpm to pellet cells.
9. Invert blocks over sink and dump media out gently. Pat gently on paper towels to remove any excess media.
10. Proceed to QIAGEN(R) prepping or cover with plate sealers and store at .20 C.