Oscarliu117 (Talk | contribs) |
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<div class="collapse multi-collapse" id="Extraction"> | <div class="collapse multi-collapse" id="Extraction"> | ||
− | <h3>Gel Dissociation</h3></br> | + | </br></br><h3>Gel Dissociation</h3></br> |
<p class="pcontent">1. Gel Extraction</br></br> | <p class="pcontent">1. Gel Extraction</br></br> | ||
a. Excised the DNA fragment from the agarose gel.</br></br> | a. Excised the DNA fragment from the agarose gel.</br></br> | ||
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</div> | </div> | ||
+ | </br> | ||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#Preparation" role="button" | ||
+ | aria-expanded="false" aria-controls="multiCollapseExample1"> | ||
+ | Competent Cell Preparation | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | |||
+ | <div class="collapse multi-collapse" id="Preparation"> | ||
+ | <p class="pcontent"></br></br>・ For E. coli DH5α,BL21(DE3) and W3100(DE3) competent cell</br></br> | ||
+ | 1. Streak out wild type E. coli on a plate (LB plate without antibiotics) overnight and pick one colony into 3 ml of media (LB) and grow overnight.</br></br> | ||
+ | 2. Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at 37 ℃.</br></br> | ||
+ | 3. When the OD600 nm up to 0.35, put the cells on ice immediately.</br></br> | ||
+ | 4. Spin the cells at 4℃for 10 minutes at 4000 rpm.</br></br> | ||
+ | 5. Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer 1(TFB1)</br></br> | ||
+ | 6. Leave nicely suspended bugs on ice for 10 minutes.</br></br> | ||
+ | 7. Spin the cells at 4℃ for 10 min. at 4000 rpm.</br></br> | ||
+ | 8. Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).</br></br> | ||
+ | 9. Leave on immediately on ice for 30 minutes.</br></br> | ||
+ | 10. Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with liquid nitrogen.</br></br> | ||
+ | 11. Store the frozen cells in the -80℃ freezer.</br></br></p> | ||
+ | |||
+ | </div> | ||
<div id="list-item-4"> | <div id="list-item-4"> |
Revision as of 15:57, 21 September 2018