Difference between revisions of "Team:Warwick/Protocols"

Line 25: Line 25:
 
<br> To ensure that our experiments went as smoothly and efficiently as possible, we followed tried and tested protocols.
 
<br> To ensure that our experiments went as smoothly and efficiently as possible, we followed tried and tested protocols.
 
</p>
 
</p>
−
<H3>Competent Cell Protocol</H3>
+
<H3>Competent Cell Protocol</H3>
 +
<h5>Produced chemically competent cells</h5>
  
  
Line 97: Line 98:
 
<br>
 
<br>
 
<br>
 
<br>
 +
 +
<h3> Chemical transformation </h3>
 +
<p> - Take chemically competent cells (-80C freezer) and thaw on ice/water mix.
 +
<br> - Add plasmid DNA to 50uL of competent cells: for minipreps 0.5-1 uL of DNA is enough, for ligations, use 5-10 uL. Mix thoroughly.
 +
<br> - Leave on ice for 30-45 minutes. Turn on the waterbath set to 42C, so it reaches the target temperature over this time.
 +
<br> - Heat shock the cells at 42C for 30 seconds. This will create pores in the competent cells through which the plasmid can enter into the cell.
 +
<br> - Put back on ice for 2 minutes. This allows the cell to recover and begin repairing the pores, preventing cell death.
 +
<br> - Add 950uL of growth media (e.g. SOC, LB, etc.) bringing the volume up to ~1000uL
 +
<br> - Incubate with shaking at 37C for 45-60 minutes.
 +
<br> - Plate 200uL on appropriate antibiotic: If using a ligation (or anything likely to have low efficiency) centrifuge the cells first at 8000rpm for 2 minutes and resuspend in 200uL of media then plate everything. If there are still some cells left after plating, the rest can be kept up to 3 weeks in a 4 degree fridge.
 +
<br> - Incubate overnight at 37C.
 +
</p>
  
 
</div>
 
</div>

Revision as of 12:49, 17 September 2015

Warwick iGEM 2015

Open Menu