Team:Oxford/protocols/Transformation into chemically competent E.coli
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<li>Heat shock the bacteria by placing them in the 42ºC water bath for NO MORE THAN 45 SECONDS. <BR><BR> | <li>Heat shock the bacteria by placing them in the 42ºC water bath for NO MORE THAN 45 SECONDS. <BR><BR> | ||
<li>Immediately place the bacteria on ICE for 1 minute. <BR><BR> | <li>Immediately place the bacteria on ICE for 1 minute. <BR><BR> | ||
- | <li>Add 800µl LB and incubate at 37ºC for 1 hour. <BR><BR> | + | <li>Add 800µl LB and incubate at 37ºC for 1 hour. <BR> |
+ | <font style="font-style:italic"NOTE: Place Eppendorfs in a Petri dish before placing in incubator.</font><BR><BR> | ||
<li>Plate the bacteria under sterile conditions (bunsen burner ON): <BR><BR> | <li>Plate the bacteria under sterile conditions (bunsen burner ON): <BR><BR> | ||
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<p>If using the C2987 cells only use 50µl aliquots as supplied. Use 2µl of assembly product (1-5µl containing 1pg-100ng of plasmid DNA).</p> | <p>If using the C2987 cells only use 50µl aliquots as supplied. Use 2µl of assembly product (1-5µl containing 1pg-100ng of plasmid DNA).</p> | ||
<p>In step 6, heat shock the bacteria by placing them in the water bath for EXACTLY 30 seconds. Leave on ice for 5 minutes.</p> | <p>In step 6, heat shock the bacteria by placing them in the water bath for EXACTLY 30 seconds. Leave on ice for 5 minutes.</p> | ||
- | + | <p>In step 8, add 950µl of SOC media and incubate as above. | |
{{:Team:Oxford/templates/footer}} | {{:Team:Oxford/templates/footer}} |
Revision as of 13:26, 23 July 2014
Transforming Competent Cells - DH5-alpha cells
↩ Back to other protocols.
- Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.
- Split the thawed cells into 2x 100µl aliquots.
- To each aliquot add 1µl of plasmid DNA.
- Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter.
- During this incubation time prepare the antibiotic agar plates:
- Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.
- Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.
- Once cool take the agar and petri dishes to the laminar flow hood.
- Add the appropriate volume of antibiotic to the agar bottle before pouring.
- Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle.
- Leave the agar plates to set (~15-30 minutes)
- Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.
- Heat shock the bacteria by placing them in the 42ºC water bath for NO MORE THAN 45 SECONDS.
- Immediately place the bacteria on ICE for 1 minute.
- Add 800µl LB and incubate at 37ºC for 1 hour.
<font style="font-style:italic"NOTE: Place Eppendorfs in a Petri dish before placing in incubator.</font>
- Plate the bacteria under sterile conditions (bunsen burner ON):
- Place a glass spreader in ethanol.
- Flame the spreader to burn off the ethanol and let it cool.
- Plate #1: spread 100µl of cell culture onto the plate
- Plate #2: spin down the remaining cells from step 3. Discard the supernatant and re-suspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step
- Place a glass spreader in ethanol.
- Incubate at 37ºC overnight.
Transforming Competent Cells - NEB aplha-5 cells
Follow the protocol as above with the following changes:
If using the C2987 cells only use 50µl aliquots as supplied. Use 2µl of assembly product (1-5µl containing 1pg-100ng of plasmid DNA).
In step 6, heat shock the bacteria by placing them in the water bath for EXACTLY 30 seconds. Leave on ice for 5 minutes.
In step 8, add 950µl of SOC media and incubate as above.