Genomic Sequencing Utilizing a RescueMu Insert:

Picking Colonies

Version #: 2

Date of last revision: 4/16/03

Author: B. Schneider

Written by: D. Morrow, B. Schneider

Stanford Genome Technology Center

Stanford University

Description:

RescueMu is transformed into DH10B E. coli (ElectroMax (BRL #18290-015)) cells that are then plated onto agar plates containing 100 ug/ml carbenicillin.  The resulting colonies are then picked into 384-well plates for subsequent use in PCR and/or storage.  This protocol outlines the picking procedure after receiving the plated RescueMu colonies.  Note: In order to ensure accurate identification of plates during genomic sequencing, barcode labeling is utilized throughout the RescueMu  protocol.  

Materials and Reagents	Vendor	Stock #
Terrific Broth (TB) Freezing Buffer*	N/A	N/A
K2HPO4 (anhydrous)	Sigma-Aldrich	P-3786
KH2PO4 (anhydrous)	Sigma-Aldrich	P-5379
C6H5Na3O7 * 2H2O	Sigma-Aldrich	S-4641
MgSO4	Sigma-Aldrich	M-7506
(NH4)2SO4	Sigma-Aldrich	A-6387
100 % Glycerol	Roche	100 649
Bacto Yeast Extract	Fisher Scientific Co.	DF0127-17-9
Bacto Yeast Extract	Fisher Scientific Co.	DF0123-17-3
Autoclaved deionized and distilled water (Autoclaved ddH2O)	Prepared at Genome Center	N/A
Ampicillin (sodium salt) (25 mg/ml)	Sigma-Aldrich	A9518
Sterile wooden toothpicks	Prepared at Genome Center	N/A
10% bleach	Prepared at Genome Center	N/A
70% ethanol	Prepared at Genome Center	N/A
Sterile picking guards for each quadrant	Custom-made at Stanford	N/A
384-well plates	Incyte Genomics	ATD-3000
Sterile, disposable Petri dish	Cole Palmer	KX-06139-02
Aluminum sealing tape	R.S. Hughes	425-3
0.45 um disposable Nalgene® Filter Unit	VWR	28199-097

*Modified LB Freezing Buffer taken from Zimmer and Verrinder Gibbins, 1997 in Molecular Cloning, third edition, Sambrook and Russell, eds.

Procedure:

Preparing TB Freezing Buffer

1. Add the following to a final volume of 100 ml autoclaved ddH2O and mix until dissolved: 
2. Add this mixture to 900 ml of TB (see below) and mix thoroughly.

Preparation of Terrific Broth (TB)

Prepared and supplied at Stanford Genome and Technology Center.

• 12 g Bacto™ Tryptone
• 24 g Bacto™ Yeast Extract
• 4 ml glycerol
• add ddH2O to a final volume of 900ml and mix
• autoclave

1. Add 40 ml of glycerol to 960 ml of the resulting solution and mix thoroughly.
2. Filter through a 0.45 um disposable Nalgene® filter.
3. Store at 4 C or room temperature.

Picking 

1. Add 600 ul of ampicillin at a concentration of 25 mg/ml in autoclaved ddH2O to 500 ml of TB Freezing Buffer  (Note: final concentration of ampicillin in the Freezing Buffer should be 30 ug/ml).  Store at 4C until ready to pick.
2. Add 65 ul of this resulting solution to each well in an Incyte Genomics 384-well plate.  Note: store any remaining TB freezing buffer with ampicillin at 4C.
3. Attach the appropriate quadrant's picking guard to the 384-well plate to be picked into.  (Since picking is done manually, picking guards are utilized to mitigate picking error and prevent cross-contamination of colonies into surrounding quadrants ((See diagram below)).  Between plates, sterilize picking guards by immersing in a 10% bleach bath for 5 minutes, rinsing briefly in ddH2O and immersing in a 70% ethanol bath for another 5 minutes.  Allow to air dry in front of a fan.)  
4. Using a sterile toothpick, scrape an individual bacterial colony from a pre-made agar plate and inoculate each well with a single colony.  Agitate by lightly tapping the toothpick up and down in the well.  Notes:  Pick only colonies without surrounding satellite colonies as they may result in poor sequencing template.  Be sure to pick into the appropriate quadrant(s) of the 384-well plate:

                   Quadrant 1                            Quadrant 2  

    Quadrant 4                                          Quadrant 3 

1. Label the side of the 384-well plate closest to the letters with the appropriate grid information for each quadrant.  Quadrants 1-4 should be labeled in consecutive order from left to right.
2. Cover the 384-well plate with the supplied plastic lid and incubate at 37 C for 17-19 hours without shaking.
3. Cover the 384-well plate with an aluminum plate seal and store at -80 C or keep unsealed on counter top at room temperature if performing PCR within a 12-hour period. 

6.271 g  K2HPO4 (anhydrous)

1.797 g  KH2PO4 (anhydrous)

0.500 g  C6H5Na3O7 * 2H2O

0.048 g  MgSO4

0.898 g  (NH4)2SO4