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| | <h2>Protocols</h2> <!--title--> | | <h2>Protocols</h2> <!--title--> |
| - | <p><h1> I. Preparation of LB Media</h1> | + | <p>help plz</p> |
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| - | <p>a. Obtain a 1 L glass bottle. </p>
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| - | <p>b. Measure out 20g of LB broth powder</p>
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| - | <p>c. Add stir bar to bottle</p>
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| - | <p>d. Add 1L of DI water to bottle</p>
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| - | <p>e. Add powder to bottle</p>
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| - | <p>f. Stir on stir plate until reasonably consistent. There should be minimal powder settled at bottom</p>
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| - | <p>g. Autoclave it</p>
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| - | <p>i. Make sure the cap is loose</p>
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| - | <p>ii. Don’t forget autoclave tape to indicate bottle reached proper temperature</p>
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| - | <p>iii. Set to 15 minutes at 121 degrees Celsius</p>
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| - | <h1> II. Creating a Glycerol Stock</h1>
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| - | <p>a. Make a stock of 50% glycerol</p>
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| - | <p>i. Obtain 2 50mL centrifuge tube</p>
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| - | <p>ii. Add 25mL 99.9% glycerol</p>
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| - | <p>iii. Add 25mL Milli-Q water</p>
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| - | <p>iv. Vortex until consistent</p>
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| - | <p>v. Filter solution through 0.22um filter using syringe into other tube</p>
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| - | <p>b. Add 560uL of overnight culture to cryogenic vial tube</p>
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| - | <p>c. Add 140uL of 50% glycerol to cryogenic vial tube</p>
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| - | <p>d. LIMS it and store in -80 degree Celsius freezer</p>
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| - | <h1>III. Gel Extraction</h1>
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| - | <p>a. Create <b>0.8%</b> Gel</p>
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| - | <p><b> i. </b>Weigh out 0.4g agarose and add to beaker</p>
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| - | <p><b> ii. </b>Measure 50mL TAE and add to beaker</p>
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| - | <p><b> iii. </b>Microwave until solution clear</p>
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| - | <p><b> iv. </b>Add 5uL Syber Safe to solution </p>
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| - | <p><b> v. </b>Pour into casting mold. Use large comb for gel extraction</p>
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| - | <p><b>b. </b>Add 10uL loading dye to 50mL digest, mix well and load.</p>
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| - | <p><b>c. </b>Run gel on 120 volts for 30 min</p>
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| - | <p><b>d. </b>Extract gel slice with clean knife, be sure to cut close to avoid excess agarose</p>
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| - | <p><b>e. </b>Weigh gel in colorless tube</p>
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| - | <p><b> i. </b>Calculate volume (100uL=100mg)</p>
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| - | <p><b>f. </b>Add 3 volumes of Buffer QG to 1 volume of gel</p>
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| - | <p><b>g. </b>Incubate at 50 degrees C for 10 min, vortex every 3 min</p>
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| - | <p><b>h. </b>Be sure solution is yellow before proceeding.</p>
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| - | <p><b>i. </b>Add 1 gel volume of isopropanol, pipet up and down</p>
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| - | <p><b>j. </b>Apply sample to QIAquick spin column</p>
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| - | <p><b>k. </b>Add 0.75 mL PE, centrifuge for 1 min</p>
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| - | <p><b>l. </b>Discard flow through, centrifuge again</p>
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| - | <p><b>m. </b>Place column into clean 1.5mL microcentrifuge tube</p>
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| - | <p><b>n. </b>Elute with 35uL of Eb</p>
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| - | <p><b>o. </b>Nanodrop for yield</p>
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| - | <h1> IV. T7 Express Induction</h1>
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| - | <p>a. Grow overnight culture of methyltransferase fusion cloned into pet26b-sgRNA-target backbone.</p>
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| - | <p>b. Measure OD on plate reader and dilute to 0.5 OD</p>
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| - | <p>c. Add IPTG at 1mM final concentration.</p>
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| - | <p>d. Grow culture in shaker incubator at 37 degrees Celsius for 5 hours.</p>
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| - | <p>e. Miniprep the culture to isolate the plasmid</p>
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| - | <h1> V. Use Our Restriction Digest Based Assay to Screen Methyltransferase-DNA Binding Domain Fusions</h1>
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| - | <p>a. Clone sequence of methyltransferase-DNA binding domain fusion into our backbone</p>
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| - | <p>b. Induce culture with IPTG according to “T7 Express Induction Protocol”</p>
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| - | <p>c. Miniprep culture</p>
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| - | <p>d. Digest 1ug of miniprep with appropriate master mix containing restriction enzymes</p>
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| - | </p> | + | |
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| | </div> | | </div> |
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