Difference between revisions of "Protocols"

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<h1>Protocols<h2>
 
<h1>Protocols<h2>
 +
<p>
 +
    Protocols
 +
</p>
 +
<p>
 +
    <strong><u>Growth media </u></strong>
 +
</p>
 +
<p>
 +
    <strong><u>LB medium </u></strong>
 +
</p>
 +
<p>
 +
    <u>Material</u>
 +
</p>
 +
<p>
 +
    2 g Tryptone
 +
</p>
 +
<p>
 +
    1 g Yeast extract
 +
</p>
 +
<p>
 +
    1 g NaCl
 +
</p>
 +
<p>
 +
    200 ml dH<sub>2</sub>O
 +
</p>
 +
<p>
 +
    <u>Method</u>
 +
</p>
 +
<p>
 +
    Using a 1L conical flask add 200ml dH2O.
 +
</p>
 +
<p>
 +
    Add 2 g Tryptone, 1 g yeast extract, 1 g NaCl to 200ml dH2O, mix until
 +
    dissolved.
 +
</p>
 +
<p>
 +
    Close with sponge and foil.
 +
</p>
 +
<p>
 +
    Autoclave<strong> </strong>125 <sup>o</sup>C/15 min
 +
</p>
 +
<p>
 +
    Store at room temperature.
 +
</p>
 +
<p>
 +
    <strong><u>LB agar</u></strong>
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    <u>Material </u>
 +
</p>
 +
<p>
 +
    500 mg Tryptone
 +
</p>
 +
<p>
 +
    250 mg Yeast extract
 +
</p>
 +
<p>
 +
    250 mg NaCl
 +
</p>
 +
<p>
 +
    750 mg Agar
 +
</p>
 +
<p>
 +
    dH<sub>2</sub>O to 50 ml
 +
</p>
 +
<p>
 +
    <u>Method</u>
 +
</p>
 +
<p>
 +
    2 x<strong> </strong>50 ml in 125 ml flask
 +
</p>
 +
<p>
 +
    Close with sponge and foil.
 +
</p>
 +
<p>
 +
    <strong>Autoclave </strong>
 +
    125 <sup>o</sup>C/15 min
 +
</p>
 +
<p>
 +
    store at room temperature.
 +
</p>
 +
<p>
 +
    <strong><u>SOC media </u></strong>
 +
</p>
 +
<p>
 +
    For 1 x 10ml solution, stored in 20ml culture tubes with a screw cap add
 +
    the following:
 +
</p>
 +
<p>
 +
    0.2 g Bacto Tryptone
 +
</p>
 +
<p>
 +
    0.05 g Bacto Yeast Extract
 +
</p>
 +
<p>
 +
    20 µl of 5M NaCl
 +
</p>
 +
<p>
 +
    25 µl of 1M KCl
 +
</p>
 +
<p>
 +
    100 µl of 1M MgCl<sub>2</sub>
 +
</p>
 +
<p>
 +
    100 µl of 1M MgSO<sub>4</sub>
 +
</p>
 +
<p>
 +
    200 µl of 1M glucose
 +
</p>
 +
<p>
 +
    Adjust to 10 ml with dH<sub>2</sub>O
 +
</p>
 +
<p>
 +
    Sterilize by autoclaving and store at room temperature.
 +
</p>
 +
<p>
 +
    <strong><u>0.1M MgCl<sub>2</sub> solution</u></strong>
 +
</p>
 +
<p>
 +
    2.033 g of MgCl<sub>2</sub> ·6H<sub>2</sub>O in 100 ml of H<sub>2</sub>O in
 +
    100 ml Pyrex bottle.
 +
</p>
 +
<p>
 +
    <strong>Autoclave</strong>
 +
    and store at 4 <sup>o</sup>C.
 +
</p>
 +
<p>
 +
    <strong><u>0.1M CaCl<sub>2</sub> solution</u></strong>
 +
</p>
 +
<p>
 +
    11 g of CaCl<sub>2</sub> ·6H<sub>2</sub>O in 500 ml of H<sub>2</sub>O in
 +
    500 ml Pyrex bottle.
 +
</p>
 +
<p>
 +
    <strong>Autoclave</strong>
 +
    and store at 4 <sup>o</sup>C.
 +
</p>
 +
<p>
 +
    <strong><u>50% glycerol solution</u></strong>
 +
</p>
 +
<p>
 +
    5 ml of glycerol and 5 ml H<sub>2</sub>O in 20 ml culture tube with screw
 +
    cup.
 +
</p>
 +
<p>
 +
    <strong>Autoclave</strong>
 +
    and store at 4 <sup>o</sup>C.
 +
</p>
 +
<p>
 +
    <strong><u>Antibiotic stock solutions </u></strong>
 +
</p>
 +
<p>
 +
    Ampicillin 100 mg/ml – 1 g in 10 ml of MilliQ water
 +
</p>
 +
<p>
 +
    Kanamycin 15 mg/ml – 0.15g in 10 ml of MilliQ water
 +
</p>
 +
<p>
 +
    Tetracycline 12.5 mg/ml – 0.125 g in 10 ml of 50% v/v ethanol
 +
</p>
 +
<p>
 +
    Chloramphenicol 34 mg/ml – 0.34 g in 10 ml of 80% v/v ethanol
 +
</p>
 +
<p>
 +
    <em>The stock solutions is filtered through a 0.2 </em>
 +
    <em>m</em>
 +
    <em>
 +
        m filter, aliquoted and stored at -20<sup>o</sup>C until use. The final
 +
        working concentration of all antibiotics is 1:1000 of the stock
 +
        solution.
 +
    </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <em> </em>
 +
</p>
 +
<p>
 +
    <strong><u>Making competent cells</u></strong>
 +
</p>
 +
<p>
 +
    <strong><u>Preparing cells</u></strong>
 +
</p>
 +
<ol>
 +
    <li>
 +
        <p>
 +
            Work under flame.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Streak 10 ml of <em>E. coli</em> cells onto one of the LB agar
 +
            plates containing no antibiotic or specific for the cell type.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Grow cell overnight at 37 <sup>o</sup>C (plate upside down), no
 +
            more than 16 h.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Wrap plate in a parafilm and store at 4 <sup>o</sup>C.
 +
        </p>
 +
        <p>
 +
            <strong><u>Making competent cells</u></strong>
 +
        </p>
 +
        <p>
 +
            <strong><u> </u></strong>
 +
        </p>
 +
    </li>
 +
</ol>
 +
<ol>
 +
    <li>
 +
        <p>
 +
            Pick single colony of the cells from the LB agar plate into 10 ml
 +
            of LB media containing no antibiotic or specific for the cell type.
 +
            Grow the cultures overnight at 37 <sup>o</sup>C with shaking at 250
 +
            rpm.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Inoculate 200 ml of prewarmed to 37<sup>o</sup>C LB medium (no
 +
            antibiotics or specific for the cell type) with 10 ml of the
 +
            overnight cultures, and grow at 37<sup>o</sup>C for 60 min, with
 +
            vigorous shaking 250 rpm or until the OD<sub>600</sub> is 0.4 -
 +
            0.5.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Put flask on ice for 30 min. At the same time chill sterile falcon
 +
            (centrifuge) tubes.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Aliquot culture into 50 ml each 4 x 50 ml chilled falcon
 +
            (centrifuge) tubes.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
Harvest the cells by centrifugation for 7 min at 3500 rpm, at 4            <sup>o</sup>C and discard supernatants completely.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Resuspend cells in each tube in 12.5 ml of 0.1 M MgCl<sub>2</sub>.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Centrifuge for 7 min at 3500 rpm, at 4<sup>o</sup>C and discard
 +
            supernatants.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Resuspend cells in each tube in 25 ml of 0.1 M CaCl<sub>2</sub>.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Incubate cells on ice for 30 min.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Centrifuge for 7 min at 3500 rpm, at 4<sup>o</sup>C and discard
 +
            supernatants.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Resuspend cells in each tube in 700 ml of 0.1 M CaCl<sub>2</sub>
 +
            and 300 ml of 50% glycerol. Final volume 1 ml in each tube.
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Aliquot 50 ml aliquots into 1.5 ml sterile microcentrifuge tube on
 +
            ice and
 +
        </p>
 +
        <p>
 +
            store at –80<sup>o</sup>C.
 +
        </p>
 +
        <p>
 +
            <strong><u>Bacterial Transformation </u></strong>
 +
        </p>
 +
    </li>
 +
</ol>
 +
<ol>
 +
    <li>
 +
        <p>
 +
            Take 50 µl of prepared <em>E. coli</em> competent cells and put on
 +
            ice for 5 min
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Add 1µl of plasmid DNA and incubate on ice for 5 min
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Heat-shock for 1 min at 42 <sup>o</sup>C
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Put on ice for 5min
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Add 250 µl of room temperature SOC buffer
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Incubate for 1 h at 37 <sup>o</sup>C with shaking at 250 rpm
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Plate on one LB agar Petri dish with appropriate antibiotic
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            Incubate at 37 <sup>o</sup>C overnight
 +
        </p>
 +
    </li>
 +
    <li>
 +
        <p>
 +
            The next day count cell colony, wrap plate in a parafilm and store
 +
            at 4 <sup>o</sup>C.
 +
        </p>
 +
        <p>
 +
            <strong><u>Spin column plasmid purification protocol</u></strong>
 +
        </p>
 +
        <p>
 +
            1. Resuspend cell pellets from 5 ml of overnight culture in 250 ml
 +
            of Buffer D1 and transfer into 1.5 ml microcentrifuge tube.
 +
        </p>
 +
        <p>
 +
            2. Add 250 ml of Buffer D2 and mix.
 +
        </p>
 +
        <p>
 +
            3. Add 350 ml of Buffer N3 and mix.
 +
        </p>
 +
        <p>
 +
            4. Centrifuge sample at 13,000 rpm for 10 min (at room
 +
            temperature).
 +
        </p>
 +
        <p>
 +
            5. Collect supernatant.
 +
        </p>
 +
        <p>
 +
            6. Place a spin column in a provided 2 ml collection tube.
 +
        </p>
 +
        <p>
 +
            7. To bind DNA, apply the sample to the column.
 +
        </p>
 +
        <p>
 +
            8. Centrifuge for 1 min. Discard flow-through and place the column
 +
            back into the same tube.
 +
        </p>
 +
        <p>
 +
            9. To wash, add 500 ml Buffer DB to the column.
 +
        </p>
 +
        <p>
 +
            10. Centrifuge for 1 min. Discard flow-through and place the column
 +
            back into the same tube.
 +
        </p>
 +
        <p>
 +
            11. To wash, add 750 ml Buffer DE to the column.
 +
        </p>
 +
        <p>
 +
            12. Centrifuge for 1 min. Discard flow-through.
 +
        </p>
 +
        <p>
 +
            13. Place the column back into the same tube.
 +
        </p>
 +
        <p>
 +
            14. Centrifuge the column for an additional 1 min and place column
 +
            in a clean 1.5 ml microcentrifuge tube.
 +
        </p>
 +
        <p>
 +
            15. To elute DNA, add 30 ml of H<sub>2</sub>O, let the column stand
 +
            for 1 min.
 +
        </p>
 +
        <p>
 +
            16. Centrifuge the column for 1 min.
 +
        </p>
 +
        <p>
 +
            17. Store purified DNA samples at -20 <sup>o</sup>C
 +
        </p>
 +
        <p>
 +
            <strong><u>Gel Electrophoresis </u></strong>
 +
        </p>
 +
        <p>
 +
            <strong><u>Stock solutions</u></strong>
 +
        </p>
 +
        <p>
 +
            <strong><u>5x TAE buffer</u></strong>
 +
        </p>
 +
        <p>
 +
            24.2 g Tris base (200mM)
 +
        </p>
 +
        <p>
 +
            5.7ml Glacial acetic acid (100mM)
 +
        </p>
 +
        <p>
 +
            3.72g Na<sub>2</sub>EDTA (5mM)
 +
        </p>
 +
        <p>
 +
            Dissolve and mix into 1L <sub> </sub>dH<sub>2</sub>O
 +
        </p>
 +
        <p>
 +
            <strong>Autoclave </strong>
 +
        </p>
 +
        <p>
 +
            Store at room temperature
 +
        </p>
 +
        <p>
 +
            <strong><u> </u></strong>
 +
        </p>
 +
        <p>
 +
            <strong><u>Agarose Gel </u></strong>
 +
        </p>
 +
    </li>
 +
</ol>
 +
<table border="1" cellspacing="0" cellpadding="0">
 +
    <tbody>
 +
        <tr>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    For ?% agarose gel
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    Agarose (g)
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    Agarose (g)
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.8%
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.24
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.48
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    1.0%
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.30
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.60
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    1.5%
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.45
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.90
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    2.0%
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    0.60
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    1.20
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    Final volume
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    30ml
 +
                </p>
 +
            </td>
 +
            <td width="200" valign="top">
 +
                <p>
 +
                    60ml
 +
                </p>
 +
            </td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
<p>
 +
    1. Into a 125 ml Erlenmeyer borosilicate glass flask weigh out and dissolve
 +
    required amount of agarose in either 30ml or 60ml 1x TAE buffer (for DNA
 +
    samples less than 20µl use 30ml of agarose. For DNA samples of 30-50µl use
 +
    60ml agarose).
 +
</p>
 +
<p>
 +
    2. Heat in microwave until dissolved (approximately 1 min at 700 w)
 +
</p>
 +
<p>
 +
    3. Cool solution to 50<sup> o</sup>C.
 +
</p>
 +
<p>
 +
    4. Add 1µl Gel Red stain to flask
 +
</p>
 +
<p>
 +
    5. Pour the gel into the prepared 7cm x 7cm gel tray with 1.5mm fixed
 +
    height comb.
 +
</p>
 +
<p>
 +
    6. Allow to set for 15-30m mins
 +
</p>
 +
<p>
 +
    7. Remove any tape from the gel mold and place the gel mold in the running
 +
    tank with the DNA samples at the cathode (black) end.
 +
</p>
 +
<p>
 +
    8. Fill the running tank with 250ml 1x TAE buffer
 +
</p>
 +
<p>
 +
    9. Mix 10µl of DNA sample with 2µl of 6x sample buffer.
 +
</p>
 +
<p>
 +
    10. Load each sample into the wells of the gel .
 +
</p>
 +
<p>
 +
    11. Run at 100V for 45-60mins
 +
</p>
 +
<p>
 +
    <strong><u>Gel analysis </u></strong>
 +
</p>
 +
<p>
 +
    If your DNA samples are intended for cloning purposes, use a long
 +
    wavelength UV light (366nm) gel viewer. Use a clean scalpel to cut the gel
 +
    with the bands present and store in a 1.5ml microcentrifuge tube for
 +
    further extraction and purification.
 +
</p>
 +
<p>
 +
    If there is no need to use the DNA for cloning purposes use the standard
 +
    short wavelength UV light (254nm) viewer.
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p align="center">
 +
    <strong> </strong>
 +
</p>
 +
<p align="center">
 +
    <strong> </strong>
 +
</p>
 +
<p align="center">
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p align="center">
 +
    <u>Spin column DNA extraction from the gel protocol</u>
 +
</p>
 +
<p>
 +
    1. Excise the DNA fragments from the gel with a clean, sharp scalpel. Weigh
 +
    the gel slice in a colorless tube. Add 3 volumes of Buffer DG to a 1 volume
 +
    of gel.
 +
</p>
 +
<p>
 +
    2. Incubate sample at 50 ­­<sup>0</sup>C for 10 min, mix by vortexing every
 +
    2-3 min.
 +
</p>
 +
<p>
 +
    3. Add 1 gel volume of isopropanol to the sample and mix.
 +
</p>
 +
<p>
 +
    4. Place a spin column in a provided 2 ml collection tube
 +
</p>
 +
<p>
 +
    5. To bind DNA, apply the sample to the column.
 +
</p>
 +
<p>
 +
    6. Centrifuge for 1 min, discard flow-through and place the column back
 +
    into the same tube
 +
</p>
 +
<p>
 +
    7. (Optional) Add 0.5 ml of Buffer DG to column and centrifuge for 1 min
 +
</p>
 +
<p>
 +
    8. Centrifuge the column for 1 min, discard flow-through and place the
 +
    column back into the same tube
 +
</p>
 +
<p>
 +
    9. To wash, add 0.75 ml Buffer DE to the column
 +
</p>
 +
<p>
 +
    10. Centrifuge for 1 min and discard flow-through.
 +
</p>
 +
<p>
 +
    11. Place the column back in the same tube.
 +
</p>
 +
<p>
 +
    12. Centrifuge the column for an additional 1 min and place column in a
 +
    clean 1.5 ml microcentrifuge tube
 +
</p>
 +
<p>
 +
    13. To elute DNA, add 30 ul of H<sub>2</sub>O and let the column stand for
 +
    1 min.
 +
</p>
 +
<p>
 +
    14. Centrifuge the column for 1 min
 +
</p>
 +
<p>
 +
    15. Store purified DNA samples at -20 <sup>o</sup>C.
 +
</p>
 +
<p>
 +
    <strong><u>Digestion </u></strong>
 +
</p>
 +
<p>
 +
    30 µl of plasmid
 +
</p>
 +
<p>
 +
    4 µl of 10x Cutsmart buffer
 +
</p>
 +
<p>
 +
    1.0 µl of Restriction enzyme
 +
</p>
 +
<p>
 +
    1.0 µl of Second restriction enzyme
 +
</p>
 +
<p>
 +
    1 µl dH<sub>2</sub>O
 +
</p>
 +
<p>
 +
    Incubate tube at 37<sup> o</sup>C for 60 minutes.
 +
</p>
 +
<p>
 +
    <strong><u>Ligation</u></strong>
 +
</p>
 +
<p>
 +
    7µl of gene
 +
</p>
 +
<p>
 +
    1 µl of plasmid
 +
</p>
 +
<p>
 +
    1 µl of 10x DNA ligase buffer
 +
</p>
 +
<p>
 +
    1 µl of 10x DNA ligase
 +
</p>
 +
<p>
 +
    Incubate at room temperature for 60 mins
 +
</p>
 +
<p>
 +
    <strong><u>PCR </u></strong>
 +
</p>
 +
<p>
 +
    <strong><u>Working solutions </u></strong>
 +
</p>
 +
<p>
 +
    10µl of forward primer + 90µl of RNase-free water
 +
</p>
 +
<p>
 +
    10µl of reverse primer + 90µl of RNase-free water
 +
</p>
 +
<p>
 +
    <strong><u>PCR mixture </u></strong>
 +
</p>
 +
<p>
 +
    In your strip PCR tubes add the following:
 +
</p>
 +
<p>
 +
    Master Mix 5µl
 +
</p>
 +
<p>
 +
    Forward primer 2.5µl
 +
</p>
 +
<p>
 +
    Reverse primer 2.5µl
 +
</p>
 +
<p>
 +
    Sample DNA 1µl
 +
</p>
 +
<p>
 +
    PCR water 14µl
 +
</p>
 +
<p>
 +
    <em>Control tube: </em>
 +
</p>
 +
<p>
 +
    Master Mix 5µl
 +
</p>
 +
<p>
 +
    Forward primer 2.5µl
 +
</p>
 +
<p>
 +
    Reverse primer 2.5µl
 +
</p>
 +
<p>
 +
    PCR water 15µl
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    PCR Machine Cycling Times and Temperatures:
 +
</p>
 +
<table border="1" cellspacing="0" cellpadding="0">
 +
    <tbody>
 +
        <tr>
 +
            <td width="179" colspan="2">
 +
                <p align="center">
 +
                    Step
 +
                </p>
 +
            </td>
 +
            <td width="120">
 +
                <p align="center">
 +
                    Temperature(<sup>o</sup>C)
 +
                </p>
 +
            </td>
 +
            <td width="104">
 +
                <p align="center">
 +
                    Time
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="179" colspan="2">
 +
                <p align="center">
 +
                    Initial Denaturation
 +
                </p>
 +
            </td>
 +
            <td width="120">
 +
                <p align="center">
 +
                    95
 +
                </p>
 +
            </td>
 +
            <td width="104">
 +
                <p align="center">
 +
                    30 sec
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="66" rowspan="3">
 +
                <p align="center">
 +
                    <br/>
 +
                    <br/>
 +
                    24 Cycles
 +
                </p>
 +
            </td>
 +
            <td width="113">
 +
                <p align="center">
 +
                    Denaturation
 +
                </p>
 +
            </td>
 +
            <td width="120">
 +
                <p align="center">
 +
                    95
 +
                </p>
 +
            </td>
 +
            <td width="104">
 +
                <p align="center">
 +
                    30 sec
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="113">
 +
                <p align="center">
 +
                    Annealing
 +
                </p>
 +
            </td>
 +
            <td width="120">
 +
                <p align="center">
 +
                    52
 +
                </p>
 +
            </td>
 +
            <td width="104">
 +
                <p align="center">
 +
                    30 sec
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="113">
 +
                <p align="center">
 +
                    Extension
 +
                </p>
 +
            </td>
 +
            <td width="120">
 +
                <p align="center">
 +
                    72
 +
                </p>
 +
            </td>
 +
            <td width="104">
 +
                <p align="center">
 +
                    30 sec
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td width="179" colspan="2">
 +
                <p align="center">
 +
                    Final Extension
 +
                </p>
 +
            </td>
 +
            <td width="120">
 +
                <p align="center">
 +
                    74
 +
                </p>
 +
            </td>
 +
            <td width="104">
 +
                <p align="center">
 +
                    5-10 min
 +
                </p>
 +
            </td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
<p>
 +
    <strong> </strong>
 +
</p>

Revision as of 20:33, 30 October 2017

Protocols

Protocols

Growth media

LB medium

Material

2 g Tryptone

1 g Yeast extract

1 g NaCl

200 ml dH2O

Method

Using a 1L conical flask add 200ml dH2O.

Add 2 g Tryptone, 1 g yeast extract, 1 g NaCl to 200ml dH2O, mix until dissolved.

Close with sponge and foil.

Autoclave 125 oC/15 min

Store at room temperature.

LB agar

Material

500 mg Tryptone

250 mg Yeast extract

250 mg NaCl

750 mg Agar

dH2O to 50 ml

Method

2 x 50 ml in 125 ml flask

Close with sponge and foil.

Autoclave 125 oC/15 min

store at room temperature.

SOC media

For 1 x 10ml solution, stored in 20ml culture tubes with a screw cap add the following:

0.2 g Bacto Tryptone

0.05 g Bacto Yeast Extract

20 µl of 5M NaCl

25 µl of 1M KCl

100 µl of 1M MgCl2

100 µl of 1M MgSO4

200 µl of 1M glucose

Adjust to 10 ml with dH2O

Sterilize by autoclaving and store at room temperature.

0.1M MgCl2 solution

2.033 g of MgCl2 ·6H2O in 100 ml of H2O in 100 ml Pyrex bottle.

Autoclave and store at 4 oC.

0.1M CaCl2 solution

11 g of CaCl2 ·6H2O in 500 ml of H2O in 500 ml Pyrex bottle.

Autoclave and store at 4 oC.

50% glycerol solution

5 ml of glycerol and 5 ml H2O in 20 ml culture tube with screw cup.

Autoclave and store at 4 oC.

Antibiotic stock solutions

Ampicillin 100 mg/ml – 1 g in 10 ml of MilliQ water

Kanamycin 15 mg/ml – 0.15g in 10 ml of MilliQ water

Tetracycline 12.5 mg/ml – 0.125 g in 10 ml of 50% v/v ethanol

Chloramphenicol 34 mg/ml – 0.34 g in 10 ml of 80% v/v ethanol

The stock solutions is filtered through a 0.2 m m filter, aliquoted and stored at -20oC until use. The final working concentration of all antibiotics is 1:1000 of the stock solution.

Making competent cells

Preparing cells

  1. Work under flame.

  2. Streak 10 ml of E. coli cells onto one of the LB agar plates containing no antibiotic or specific for the cell type.

  3. Grow cell overnight at 37 oC (plate upside down), no more than 16 h.

  4. Wrap plate in a parafilm and store at 4 oC.

    Making competent cells

  1. Pick single colony of the cells from the LB agar plate into 10 ml of LB media containing no antibiotic or specific for the cell type. Grow the cultures overnight at 37 oC with shaking at 250 rpm.

  2. Inoculate 200 ml of prewarmed to 37oC LB medium (no antibiotics or specific for the cell type) with 10 ml of the overnight cultures, and grow at 37oC for 60 min, with vigorous shaking 250 rpm or until the OD600 is 0.4 - 0.5.

  3. Put flask on ice for 30 min. At the same time chill sterile falcon (centrifuge) tubes.

  4. Aliquot culture into 50 ml each 4 x 50 ml chilled falcon (centrifuge) tubes.

  5. Harvest the cells by centrifugation for 7 min at 3500 rpm, at 4 oC and discard supernatants completely.

  6. Resuspend cells in each tube in 12.5 ml of 0.1 M MgCl2.

  7. Centrifuge for 7 min at 3500 rpm, at 4oC and discard supernatants.

  8. Resuspend cells in each tube in 25 ml of 0.1 M CaCl2.

  9. Incubate cells on ice for 30 min.

  10. Centrifuge for 7 min at 3500 rpm, at 4oC and discard supernatants.

  11. Resuspend cells in each tube in 700 ml of 0.1 M CaCl2 and 300 ml of 50% glycerol. Final volume 1 ml in each tube.

  12. Aliquot 50 ml aliquots into 1.5 ml sterile microcentrifuge tube on ice and

    store at –80oC.

    Bacterial Transformation

  1. Take 50 µl of prepared E. coli competent cells and put on ice for 5 min

  2. Add 1µl of plasmid DNA and incubate on ice for 5 min

  3. Heat-shock for 1 min at 42 oC

  4. Put on ice for 5min

  5. Add 250 µl of room temperature SOC buffer

  6. Incubate for 1 h at 37 oC with shaking at 250 rpm

  7. Plate on one LB agar Petri dish with appropriate antibiotic

  8. Incubate at 37 oC overnight

  9. The next day count cell colony, wrap plate in a parafilm and store at 4 oC.

    Spin column plasmid purification protocol

    1. Resuspend cell pellets from 5 ml of overnight culture in 250 ml of Buffer D1 and transfer into 1.5 ml microcentrifuge tube.

    2. Add 250 ml of Buffer D2 and mix.

    3. Add 350 ml of Buffer N3 and mix.

    4. Centrifuge sample at 13,000 rpm for 10 min (at room temperature).

    5. Collect supernatant.

    6. Place a spin column in a provided 2 ml collection tube.

    7. To bind DNA, apply the sample to the column.

    8. Centrifuge for 1 min. Discard flow-through and place the column back into the same tube.

    9. To wash, add 500 ml Buffer DB to the column.

    10. Centrifuge for 1 min. Discard flow-through and place the column back into the same tube.

    11. To wash, add 750 ml Buffer DE to the column.

    12. Centrifuge for 1 min. Discard flow-through.

    13. Place the column back into the same tube.

    14. Centrifuge the column for an additional 1 min and place column in a clean 1.5 ml microcentrifuge tube.

    15. To elute DNA, add 30 ml of H2O, let the column stand for 1 min.

    16. Centrifuge the column for 1 min.

    17. Store purified DNA samples at -20 oC

    Gel Electrophoresis

    Stock solutions

    5x TAE buffer

    24.2 g Tris base (200mM)

    5.7ml Glacial acetic acid (100mM)

    3.72g Na2EDTA (5mM)

    Dissolve and mix into 1L dH2O

    Autoclave

    Store at room temperature

    Agarose Gel

<tbody> </tbody>

For ?% agarose gel

Agarose (g)

Agarose (g)

0.8%

0.24

0.48

1.0%

0.30

0.60

1.5%

0.45

0.90

2.0%

0.60

1.20

Final volume

30ml

60ml

1. Into a 125 ml Erlenmeyer borosilicate glass flask weigh out and dissolve required amount of agarose in either 30ml or 60ml 1x TAE buffer (for DNA samples less than 20µl use 30ml of agarose. For DNA samples of 30-50µl use 60ml agarose).

2. Heat in microwave until dissolved (approximately 1 min at 700 w)

3. Cool solution to 50 oC.

4. Add 1µl Gel Red stain to flask

5. Pour the gel into the prepared 7cm x 7cm gel tray with 1.5mm fixed height comb.

6. Allow to set for 15-30m mins

7. Remove any tape from the gel mold and place the gel mold in the running tank with the DNA samples at the cathode (black) end.

8. Fill the running tank with 250ml 1x TAE buffer

9. Mix 10µl of DNA sample with 2µl of 6x sample buffer.

10. Load each sample into the wells of the gel .

11. Run at 100V for 45-60mins

Gel analysis

If your DNA samples are intended for cloning purposes, use a long wavelength UV light (366nm) gel viewer. Use a clean scalpel to cut the gel with the bands present and store in a 1.5ml microcentrifuge tube for further extraction and purification.

If there is no need to use the DNA for cloning purposes use the standard short wavelength UV light (254nm) viewer.

Spin column DNA extraction from the gel protocol

1. Excise the DNA fragments from the gel with a clean, sharp scalpel. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer DG to a 1 volume of gel.

2. Incubate sample at 50 ­­0C for 10 min, mix by vortexing every 2-3 min.

3. Add 1 gel volume of isopropanol to the sample and mix.

4. Place a spin column in a provided 2 ml collection tube

5. To bind DNA, apply the sample to the column.

6. Centrifuge for 1 min, discard flow-through and place the column back into the same tube

7. (Optional) Add 0.5 ml of Buffer DG to column and centrifuge for 1 min

8. Centrifuge the column for 1 min, discard flow-through and place the column back into the same tube

9. To wash, add 0.75 ml Buffer DE to the column

10. Centrifuge for 1 min and discard flow-through.

11. Place the column back in the same tube.

12. Centrifuge the column for an additional 1 min and place column in a clean 1.5 ml microcentrifuge tube

13. To elute DNA, add 30 ul of H2O and let the column stand for 1 min.

14. Centrifuge the column for 1 min

15. Store purified DNA samples at -20 oC.

Digestion

30 µl of plasmid

4 µl of 10x Cutsmart buffer

1.0 µl of Restriction enzyme

1.0 µl of Second restriction enzyme

1 µl dH2O

Incubate tube at 37 oC for 60 minutes.

Ligation

7µl of gene

1 µl of plasmid

1 µl of 10x DNA ligase buffer

1 µl of 10x DNA ligase

Incubate at room temperature for 60 mins

PCR

Working solutions

10µl of forward primer + 90µl of RNase-free water

10µl of reverse primer + 90µl of RNase-free water

PCR mixture

In your strip PCR tubes add the following:

Master Mix 5µl

Forward primer 2.5µl

Reverse primer 2.5µl

Sample DNA 1µl

PCR water 14µl

Control tube:

Master Mix 5µl

Forward primer 2.5µl

Reverse primer 2.5µl

PCR water 15µl

PCR Machine Cycling Times and Temperatures:

<tbody> </tbody>

Step

Temperature(oC)

Time

Initial Denaturation

95

30 sec



24 Cycles

Denaturation

95

30 sec

Annealing

52

30 sec

Extension

72

30 sec

Final Extension

74

5-10 min