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<input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (yeast)" /> | <input type="button" style="height:50px; width:50%; BACKGROUND-COLOR: #3399FF; font-size:25; color:black;" onclick="showSpoiler(this);" value="Transformation (yeast)" /> | ||
<div class="inner" style="display:none;"> | <div class="inner" style="display:none;"> | ||
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<b>1.Preparation of Competent Cells </b> | <b>1.Preparation of Competent Cells </b> | ||
<br> <br> | <br> <br> | ||
− | <div | + | <div style="text-align: justify; margin-left:40px">1.Grow yeast cells at 30 °C in 10 ml YPD broth until mid-log phase <br>(~5 x 10<sup>6</sup> - 2 x 10<sup>7</sup> cells/ml or OD 600 of 0.8-1.0). The following steps are accomplished at room temperature. </div> |
<div style="text-align: justify; margin-left:40px"> | <div style="text-align: justify; margin-left:40px"> | ||
− | + | 2. Pellet the cells at 500 x g for 4 minutes and discard the supernatant. <br> | |
− | + | 3. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant. <br> | |
− | + | 4. Add 1 ml EZ 2 solution to resuspend the pellet. At this point, the competent cells can be used for transformations directly or stored frozen at or below -70°C for future use. It is important to freeze the cells slowly. To accomplish this, either wrap the aliquotted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer.DO NOT use liquid nitrogen to snap-freeze the cells. </div><br> | |
<b>2. Transformation </b> | <b>2. Transformation </b> | ||
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+ | <div style="text-align: justify; margin-left:20px"> | ||
This part of the procedure is the same for both frozen stored (thawed at room temperature) and freshly prepared competent yeast cells. </div> | This part of the procedure is the same for both frozen stored (thawed at room temperature) and freshly prepared competent yeast cells. </div> | ||
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− | 1. Mix 50 μl of competent cells with 0.2-1 μg DNA (in less than 5 μl volume); add 500 μl EZ 3 solution and mix thoroughly. < | + | 1. Mix 50 μl of competent cells with 0.2-1 μg DNA (in less than 5 μl volume); add 500 μl EZ 3 solution and mix thoroughly. <br> |
− | + | 2. Incubate at 30 °C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation. <br> | |
− | 2. Incubate at 30 °C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation. < | + | |
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3. Spread 50-150 | 3. Spread 50-150 | ||
μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants. </div> | μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants. </div> | ||
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Revision as of 09:28, 30 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols
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