Line 265: | Line 265: | ||
<div id="P6-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P6"> | <div id="P6-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P6"> | ||
<div class="panel-body"> | <div class="panel-body"> | ||
− | |||
<ol> | <ol> | ||
<li>Pick single colony of transformed C41 cells to 5ml LB solution with 1x antibiotics to grow starter. <br></li> | <li>Pick single colony of transformed C41 cells to 5ml LB solution with 1x antibiotics to grow starter. <br></li> | ||
<li>1% Inoculation in two 1L conical flask, each with 250 ml 2XYT solution 1x antibiotics overnight. </li> | <li>1% Inoculation in two 1L conical flask, each with 250 ml 2XYT solution 1x antibiotics overnight. </li> | ||
− | <li>Spin down 100ml cells in 50 ml falcon. Discard the supernatant. | + | <li>Spin down 100ml cells in 50 ml falcon. Discard the supernatant.</li> |
− | </li> | + | |
<li>Wash cell pellet with 40 ml cool TE buffer.</li> | <li>Wash cell pellet with 40 ml cool TE buffer.</li> | ||
− | <li>Re-suspend cells with cold 15 ml Protein Lysis Buffer (PLB). | + | <li>Re-suspend cells with cold 15 ml Protein Lysis Buffer (PLB). </li> |
− | + | <li>Sonicate on ice.</li> | |
− | + | <li>Spin at 4°C at 13000 rpm for 5 min</li> | |
− | </li> | + | <li>Collect the supernatant and dialysis overnight.</li> |
− | <li>Sonicate on ice. | + | |
− | </li> | + | |
− | <li>Spin at 4°C at 13000 rpm for 5 min | + | |
− | </li> | + | |
− | <li>Collect the supernatant and dialysis overnight. | + | |
− | + | ||
<li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | <li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | ||
− | <li>Purify proteins with ion exchange column using start buffer (20 mM Tris-HCl, pH 8.0) | + | <li>Purify proteins with ion exchange column using start buffer (20 mM Tris-HCl, pH 8.0)</li> |
− | </li> | + | <li>Apply the sample at 5 ml/min for 5 ml columns by pumping it onto the column.</li> |
− | <li>Apply the sample at 5 ml/min for 5 ml columns by pumping it onto the column. | + | <li>Trace the proteins by naked eyes.</li> |
− | </li> | + | |
− | <li>Trace the proteins by naked eyes. | + | |
− | </li> | + | |
<li>Re-suspend cells by light vortexing.</li> | <li>Re-suspend cells by light vortexing.</li> | ||
<li>Elute amajLime and mRFP at 0 µM and 30 µM NaCl respectively.</li> | <li>Elute amajLime and mRFP at 0 µM and 30 µM NaCl respectively.</li> | ||
Line 294: | Line 283: | ||
<li>After the completed elution, regenerate the column by washing with 5 column volumes of regeneration buffer.</li> | <li>After the completed elution, regenerate the column by washing with 5 column volumes of regeneration buffer.</li> | ||
<li>Remove the female luer for buffer running through the column to the waste collection tube.</li> | <li>Remove the female luer for buffer running through the column to the waste collection tube.</li> | ||
− | <li>Mix 1 volume of protein sample to 1 volume of Binding Buffer (~200 µl). | + | <li>Mix 1 volume of protein sample to 1 volume of Binding Buffer (~200 µl).</li> |
− | </li> | + | <li>Transfer all 400 µl of protein/Binding Buffer mix to the column carefully without disturbing the top of the bed containing the settled HIC matrix.</li> |
− | <li>Transfer all 400 µl of protein/Binding Buffer mix to the column carefully without disturbing the top of the bed containing the settled HIC matrix. | + | |
− | </li> | + | |
<li>Trace the fluorescent protein by naked eyes or blue light.</li> | <li>Trace the fluorescent protein by naked eyes or blue light.</li> | ||
<li>Wash the resin with 1 ml Wash buffer when the meniscus reaches the top of the bed and the run-through is collected in the waste collection tube.</li> | <li>Wash the resin with 1 ml Wash buffer when the meniscus reaches the top of the bed and the run-through is collected in the waste collection tube.</li> | ||
− | <li>Add 1 ml TE buffer to the resin and the run-through without fluorescent proteins is collected in the waste collection tube. | + | <li>Add 1 ml TE buffer to the resin and the run-through without fluorescent proteins is collected in the waste collection tube.</li> |
− | </li> | + | <li>Continue adding TE buffer to run through fluorescent proteins and collect with Eppendorf.</li> |
− | <li>Continue adding TE buffer to run through fluorescent proteins and collect with Eppendorf. | + | |
− | </li> | + | |
<li>Run SDS-PAGE to determine purity.</li> | <li>Run SDS-PAGE to determine purity.</li> | ||
− | <li>Determine protein concentration by refractometer. | + | <li>Determine protein concentration by refractometer.</li> |
− | </li> | + | |
</ol> | </ol> | ||
Line 329: | Line 313: | ||
<div id="P7-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P7"> | <div id="P7-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P7"> | ||
<div class="panel-body"> | <div class="panel-body"> | ||
− | |||
<ol> | <ol> | ||
<li>Harvest 3 mL cells at 1OD. <br></li> | <li>Harvest 3 mL cells at 1OD. <br></li> | ||
<li>Centrifuge at 14,000 rpm for 30 s. </li> | <li>Centrifuge at 14,000 rpm for 30 s. </li> | ||
− | <li>Discard the supernatant and resuspend the pellet with remnant. | + | <li>Discard the supernatant and resuspend the pellet with remnant.</li> |
− | </li> | + | |
<li>Resuspend with 250 μl Resuspension Buffer.</li> | <li>Resuspend with 250 μl Resuspension Buffer.</li> | ||
<li>Add 250 μl Lysis Buffer. </li> | <li>Add 250 μl Lysis Buffer. </li> | ||
− | <li>Add 350 μl Neutralization Buffer. | + | <li>Add 350 μl Neutralization Buffer.</li> |
− | </li> | + | <li>Centrifuge at 13,000 rpm for 10 mins.</li> |
− | <li>Centrifuge at 13,000 rpm for 10 mins. | + | <li>Transfer supernatent to column.</li> |
− | </li> | + | |
− | <li>Transfer supernatent to column. | + | |
− | + | ||
<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
− | <li>Add 700 μl Washing Buffer B. | + | <li>Add 700 μl Washing Buffer B.</li> |
− | </li> | + | |
<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
<li>Column dring with centrifuge 13.000rpm for 1 min.</li> | <li>Column dring with centrifuge 13.000rpm for 1 min.</li> | ||
Line 403: | Line 381: | ||
<li>Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm. </li> | <li>Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm. </li> | ||
<li>Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h) </li> | <li>Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h) </li> | ||
− | <li>While the cell is growing, mixing the the following reagents to be the Ca/glycerol buffer. | + | <li>While the cell is growing, mixing the the following reagents to be the Ca/glycerol buffer. </li> |
− | + | <li>Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes. </li> | |
− | </li> | + | <li>Collect the cell by centrifuging at 1000 g for 10 min at 4°C. </li> |
− | <li>Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes. | + | <li>Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. * Cells must remain clod for the rest of the procedures! </li> |
− | + | ||
− | + | ||
− | <li>Collect the cell by centrifuging at 1000 g for 10 min at 4°C. | + | |
− | </li> | + | |
− | <li> | + | |
− | </li> | + | |
<li>Collect the cell by centrifuging at 1000 g for 10 min at 4 °C. </li> | <li>Collect the cell by centrifuging at 1000 g for 10 min at 4 °C. </li> | ||
<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
Line 430: | Line 402: | ||
</div> | </div> | ||
<div class="some-padding"></div> | <div class="some-padding"></div> | ||
− | + | <div class="some-padding"></div> | |
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
Line 444: | Line 416: | ||
</div> | </div> | ||
<div id="P0-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P0"> | <div id="P0-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P0"> | ||
− | <div class="panel-body"> | + | <div class="panel-body"> |
− | + | ||
<ol> | <ol> | ||
<li>Set up PCR mixture as follow :</li> | <li>Set up PCR mixture as follow :</li> | ||
Line 489: | Line 460: | ||
<th>Cycle</th> | <th>Cycle</th> | ||
</tr> | </tr> | ||
− | + | <li>Set up thermocycler for PCR reaction as follow:</li> | |
− | + | <li>Add the DNase-free ddH2O in PCR tube. <br></li> | |
<tr> | <tr> | ||
<td>Initial denaturation </td> | <td>Initial denaturation </td> |
Revision as of 17:05, 22 August 2017