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From 2010.igem.org

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Revision as of 19:40, 21 October 2010

Using openwetware.org protocol

we wil be using restriction enzymes to peice biobrick T9002 purified DNA with LacZ pruified DNA.

Cutting LacZ @ or with SP and T9002.

a) + E,X creating EX-|__|-SP see assembly pg on IGem website. the Protocol/Restriction Digest.

Today I electorporated EColi w/part sbia3 which is TCN resistant plasmid following protocol pg 15 after electroporation I placed into incubator at 37C over night (using Top 10 electrocomp cells)

Electroporation restults-- 1.8 kv@ 5.8ms. Both pulses same number.

Assay for B-Gal activity

1. thaw Top 10 cells in 10 ml TSB broth

2. A600 of .5ml/500 ul

3. Incubate @ 37C.

4. Add a loopful of 40 ml TSB @ 23C for Monday

Objective -- grow E.Coli in absence of lactos

add lactose to induce lac operon [This will model out induction of B-Gal by AHL] measure B-gal expression by conversin of substrate ONPG (yellow color)

-after plating last night the SB1A3 spl on TCN/LB-Agar no growth appeared so, now we are going to use a lover con of TCN in two new plates then restreak and let grow over night

Contents 1 B-galactosidase Assay 1.1 Before Starting 1.2 Sample Preparation 1.3 B-galactosidase Assay

B-galactosidase Assay

Before Starting

Be sum to have cells trunsfected with a lacZ construct and prepare the following solutions:

• Dilute the 10X PBS and 10X Cleavage Buffer to make IX solutions by adding 90 ml

distilled, deionized water to 10 ml of stock solution.

• Add 270 ul B-mereaptoethanol to 100 ml IX Cleavage ButTer before use.

• Unused IX solutions may be stored at +4C for 6 months for use in future essays.

Sample Preparation

1. Starting with transfected cells, remove the growth medium from the cells and wash transfected cell monolayers once with 1X PBS.

2. Harvest cell monolayers with trypsin/EDTA or by scraping cells into 1 ml 1X PBS.

3. Centrifuge cells at 250 x g for 5 minutes. Aspirate the supernatant

4. Completely resuspend the pellet in IX Lysis Buffer. Keep sample at +4°C. Note: The amount of Lysis Buffer used varies depending on the size of the cell pellet. For a pellet harvested 48-72 hours posttransfection from a 60 mm plate, use 50 ul IX Lysis Buffer. For a 100 mm plate, use 100 pL

4. Freeze the sample on dry, ice and thaw in a 37°C water bath. Repeat 2 times.

5. Pellet the insoluble cell material by eentrifugation at maximum speed at +4°C fur 5 minutes. Transfer the supernatant to a new micrecentdfuge tube.

B-galactosidase Assay

1. For each sample (see Step 5 above), take 1-10 pl of cell lysate and transfer to a fresh microcentrifilge tube.

2. Bring to a final volume of 30 lal with distilled, deionized water.

3. Add 70 til of ONPG and 200 Ill IX Cleavage Buffer with [~-mereaptoethanol. Mix by gently flicking the tube and centri~ging briefly.

4. Incubate the tube at 37°C for 30 minutes. You should see a faint yellow color develop if I]-galactosidase is prasenL

5. To stop Ihe reaction, add 500 pl of Stop Buffer. There may be some intensifying of the color. Final volume is 800

6. Read the absorbance at 420 nm against a blank containing ONPG and Cleavage Buffer without lysatu. Be sure to assay a sample of the unlransfeeted cell lysate as a control.

7. Assay at least three different volumes oflysate (i.e. 1, 5, and 10 pl). Changes in absorbance should be linear with respect to the amount oflysate assayed. If it is not, you will not get an accumta determination of activity.

8. Once you bare obtained an accurate reading of your lysate, determine the protein concentration of the lysate, and calculate the specific activity of the lysate using the following formula:

Specific activity = nmoles of ONPG hydrolyzed/t/mg protein nmoles ofONPG bydrolyzed =(OD+20) (8 x 10~ nanoliters)/(4500 ul/nnoles-cm)(l cm)

where 4500 is the extinction coefficient, t = the time of incubation in minutes at 37°C (i.e. 30 minutes), and mg protein is the amount of protein assayed, which can be determined using the BCA assay (Pierce Chemical). Be sure to subtract the background activity of the uniransrected cell lysate. --JHullRchamberlin 19:40, 21 October 2010 (UTC)