Difference between revisions of "Team:NYU Shanghai/Protocols"

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   Estimated time: 3 hours (plus 14-18 hour incubation)
 
   Estimated time: 3 hours (plus 14-18 hour incubation)
 
   <br><br>Materials
 
   <br><br>Materials
−
     <li>2µl resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)
+
     <li>2µl resuspended DNA
 
     <li>Competent cells (50ul per transformation)
 
     <li>Competent cells (50ul per transformation)
−
     <li>Ice (in ice bucket/container)
+
     <li>Ice
−
     <li>2ml tube (1 per a transformation')
+
     <li>2ml tube
 
     <li>42ºC water bath
 
     <li>42ºC water bath
−
     <li>SOC media
+
     <li>SOC or LB media
−
     <li>1 small LB plate and 1 large LB+antibiotic per transformation
+
     <li>1 LB plate and 1 LB+antibiotic per transformation
 
     <li>Spreader
 
     <li>Spreader
 
     <li>37ºC incubator
 
     <li>37ºC incubator
 +
    <li>Shaker
 
   <br><br>Procedure
 
   <br><br>Procedure
 
   <ol>
 
   <ol>
−
     <li>Start thawing the competent cells on ice.</li>
+
     <li>Thaw competent cells on ice.</li>
−
     <li>Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.</li>
+
     <li>Add 50µL of thawed competent cells into pre-chilled 2ml tube.</li>
−
     <li>Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.</li>
+
     <li>Add 2µL of resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Keep the competent cells on ice.</li>
 
     <li>Close tubes and incubate the cells on ice for 30 minutes.</li>
 
     <li>Close tubes and incubate the cells on ice for 30 minutes.</li>
 
     <li>Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.</li>
 
     <li>Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.</li>
 
     <li>Incubate the cells on ice for 5 minutes.</li>
 
     <li>Incubate the cells on ice for 5 minutes.</li>
−
     <li>Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation</li>
+
     <li>Add 200 μl of SOC or LB media to each transformation</li>
−
     <li>Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.</li>
+
     <li>Incubate the cells at 37ºC for 2 hours at 260rpm in the shaker.</li>
−
     <li>Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 200 µl of the transformation onto the labeled antibiotic dishes, and spread. This helps ensure that you will be able to pick out a single colony. </li>
+
     <li>Label petri dishes with the appropriate antibiotic with the part number, plasmid backbone, antibiotic resistance, and date. Plate 200 µl of the transformation onto the labeled antibiotic dishes, and spread. This helps ensure that you will be able to pick out a single colony. </li>
 
     <li>Spread remaining transformation onto labeled LB agar plate and spread, approx 50µl.</li>
 
     <li>Spread remaining transformation onto labeled LB agar plate and spread, approx 50µl.</li>
 
     <li>Incubate the plates at 37ºC for 12-18 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.</li>
 
     <li>Incubate the plates at 37ºC for 12-18 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.</li>
 
     <li>You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.</li>
 
     <li>You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.</li>
 +
  <br><br>Controls
 +
    <li>Competent cells on LB plate
 +
    <li>Competent cells on LB + antibiotic plate
 +
    <li>Competent cells + plasmid on LB plate
 
   </div>
 
   </div>
 
</div>
 
</div>

Revision as of 19:45, 11 September 2015

Protocols

Recipes

3A Assembly

Making Color