Wolf S B-Gal Assay for Bacteria

Wolf’s B-gal assay for Bacteria

Growing up the cells  Grow up the cells in test tubes as normal overnight or in block (inoculate 4 colonies in a single column)  Transfer 30ul of overnight culture to a second block with 1ml media and incubate 3hrs1  To determine the culture density, dilute 50ul of culture from the block and add 150ul of sterile water (1:4 dilution) and measure OD600.

 Once correct OD600 is reached, place the blocks on ice for 30min.

Permeabilization of cells  In 2 times as many blocks used in ‘growing up the cells’1, add 1ml of Z buffer (volume depends on volume of induced culture added) and 25ul of fresh 0.1% SDS and 50ul of chloroform into each well.  Transfer 50-500ul from induced culture to another block where there will be 2 for every 1 transferred from the previous block. So if 5A/9G+IPTG1 will become 5A/9G+IPTG1A and 5A/9G+IPTG1B.  Permeabilize the bacteria cells by pumping up and down 10-15 times with pipettor.

1 See sample sheet

Created by Dwayne Taliaferro…If this doesn’t work for you feel free to try something else. Assay of b-gal activity  Allow the chloroform to settle to the bottom of the wells of the block  Transfer 100ul aliquots of permeabilized cells to microplates.  Initiate the reaction at time zero by adding 25ul of 4mg/ml ONPG2 to each well.  Incubate the reaction until post it yellow or yellow tip yellow is achieved (~10min).

 Terminate the reaction using 50ul of 1M Na2CO3.

 Measure the OD420, and OD550 and corresponding OD600 data to compute miller units.

Recipe for Z Buffer (1 liter): 16.1g Na2HPO4-7H2O 5.5g NaH2PO4-H2O 0.75g KCl 0.246g MgSO4-7H2O 2.7ml β-mercaptoethanol or can add 270ul/100ml H2O to 1 liter (filter sterilize and store at room temperature)

2 Also known as 2-nitrophenyl β-D-galactopyranoside

Created by Dwayne Taliaferro…If this doesn’t work for you feel free to try something else.