Protocols

I. Preparation of LB Media

a. Obtain a 1 L glass bottle.

b. Measure out 20g of LB broth powder

c. Add stir bar to bottle

d. Add 1L of DI water to bottle

e. Add powder to bottle

f. Stir on stir plate until reasonably consistent. There should be minimal powder settled at bottom

g. Autoclave it

i. Make sure the cap is loose

ii. Don’t forget autoclave tape to indicate bottle reached proper temperature

iii. Set to 15 minutes at 121 degrees Celsius

II. Creating a Glycerol Stock

a. Make a stock of 50% glycerol

i. Obtain 2 50mL centrifuge tube

ii. Add 25mL 99.9% glycerol

iii. Add 25mL Milli-Q water

iv. Vortex until consistent

v. Filter solution through 0.22um filter using syringe into other tube

b. Add 560uL of overnight culture to cryogenic vial tube

c. Add 140uL of 50% glycerol to cryogenic vial tube

d. LIMS it and store in -80 degree Celsius freezer

III. Gel Extraction

a. Create 0.8% Gel

i. Weigh out 0.4g agarose and add to beaker

ii. Measure 50mL TAE and add to beaker

iii. Microwave until solution clear

iv. Add 5uL Syber Safe to solution

v. Pour into casting mold. Use large comb for gel extraction

b. Add 10uL loading dye to 50mL digest, mix well and load.

c. Run gel on 120 volts for 30 min

d. Extract gel slice with clean knife, be sure to cut close to avoid excess agarose

e. Weigh gel in colorless tube

i. Calculate volume (100uL=100mg)

f. Add 3 volumes of Buffer QG to 1 volume of gel

g. Incubate at 50 degrees C for 10 min, vortex every 3 min

h. Be sure solution is yellow before proceeding.

i. Add 1 gel volume of isopropanol, pipet up and down

j. Apply sample to QIAquick spin column

k. Add 0.75 mL PE, centrifuge for 1 min

l. Discard flow through, centrifuge again

m. Place column into clean 1.5mL microcentrifuge tube

n. Elute with 35uL of Eb

o. Nanodrop for yield

IV. T7 Express Induction

a. Grow overnight culture of methyltransferase fusion cloned into pet26b-sgRNA-target backbone.

b. Measure OD on plate reader and dilute to 0.5 OD

c. Add IPTG at 1mM final concentration.

d. Grow culture in shaker incubator at 37 degrees Celsius for 5 hours.

e. Miniprep the culture to isolate the plasmid

V. Use Our Restriction Digest Based Assay to Screen Methyltransferase-DNA Binding Domain Fusions

a. Clone sequence of methyltransferase-DNA binding domain fusion into our backbone

b. Induce culture with IPTG according to “T7 Express Induction Protocol”

c. Miniprep culture

d. Digest 1ug of miniprep with appropriate master mix containing restriction enzymes