Team:Caltech/Protocols

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Revision as of 23:27, 23 June 2011

Caltech iGEM 2011

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Transforming DNA from Distribution Plates:
1) Thaw competent cells on ice. 2) Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.
3) Transfer into storage tube.
4) Pipette 1-2 microliters of the DNA into the competent cell tubes.
7) Stir with pipette tip, gently flick tube.
8) Leave on ice for 30 minutes.
9) Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.
10) Pipette 500 micro Liters of S.O.C. (LB + glucose) into each competent cell tube and incubate for 0-60 minutes before plating.

Enrichment cultures

For BPA (since soluble)

1) Set up 16 tubes: 8 tubes with vitamin media vs. 8 tubes with media (no vitamin), 4 tubes each of the four locations.
2) Place 8 test tubes in 30°C shaker and 8 test tubes in room temperature shaker.

For 17α-estradiol, bisphenol A, and nonylphenol (since non-soluble)

1) Set up two flasks: one with vitamin media, one without vitamin.
2) Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.

@@ Line 3: / Line 3: @@
 [[Team:Caltech/Recipes|Recipes for Mixes]] . [[Team:Caltech/Basicprep|Basic Preparation]]
-<p>'''Transforming DNA:'''<br/>
+<p>'''Transforming DNA from Distribution Plates:'''<br/>
-) Thaw competent cells on ice: 15K, 15J, 15O, 15M.<br/>
+) Thaw competent cells on ice.
-) Get 10 micro Liters of pure water.<br/>
+) Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.<br/>
-) Pipette out DNA from the source plate and put it in the appropriate tubes.<br/>
+) Transfer into storage tube.<br/>
-) Flick the competent cells in the tube gently.<br/>
+) Pipette 1-2 microliters of the DNA into the competent cell tubes.<br/>
-) Pipette 40 micro Liters of competent cells into the DNA.<br/>
+) Stir with pipette tip, gently flick tube.<br/>
-) Pipette 2 micro Liters of the DNA and put it in the competent cell tubes.<br/>
-) Stir a bit.<br/>
 ) Leave on ice for 30 minutes.<br/>
-) Heat Shock for 45 sec by using a water bath set to 42°C and then thawing on ice for 2 min.<br/>
+) Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.<br/>
-) Pipette 500 micro Liters of S.O.C. (LB + glucose) into each competent cell tubes.<br/>  </p>
+) Pipette 500 micro Liters of S.O.C. (LB + glucose) into each competent cell tube and incubate for 0-60 minutes before plating.<br/>  </p>
 <p>'''Enrichment cultures'''<br/>