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Protocol/1
From 2010.igem.org
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| Line 24: | Line 24: | ||
Procedure: | Procedure: | ||
| - | * Chill a box of pipette tips at -4 C and put your reaction tubes on ice. | + | * Chill a box of pipette tips at -4<sup>o</sup>C and put your reaction tubes on ice. |
| - | * Obtain DH5α cells from the –80 C freezer and thaw in palm. Store on ice for 10 minutes. | + | * Obtain DH5α cells from the –80<sup>o</sup>C freezer and thaw in palm. Store on ice for 10 minutes. |
* Use a chilled tip to transfer 100 ul of the DH5α cells into each tube. Set aside one tube as a control. | * Use a chilled tip to transfer 100 ul of the DH5α cells into each tube. Set aside one tube as a control. | ||
* Pipet up to 5uL of the plasmid (up to 50ng per 100ul of competent cells, plasmid amount. Should not exceed 5% that of the competent cells) into each tube. Swirl. | * Pipet up to 5uL of the plasmid (up to 50ng per 100ul of competent cells, plasmid amount. Should not exceed 5% that of the competent cells) into each tube. Swirl. | ||
* Store on ice for 30 minutes. | * Store on ice for 30 minutes. | ||
| - | * Place tubes in incubator set to 42<sup> | + | * Place tubes in incubator set to 42<sup>o</sup>C for EXACTLY 90 seconds. |
* Return the tubes to ice for 2 minutes. | * Return the tubes to ice for 2 minutes. | ||
* Add 800uL of LB to each tube. | * Add 800uL of LB to each tube. | ||
Revision as of 01:09, 26 October 2010
Protocol 1: Transformations
Reagents:
- Competent DH5α cells (100uL per transformation)
- up to 5uL of plasmid you want to transform
- 800uL of LB (non-contaminated) per transformation
- Plate with correct antibiotics
Procedure:
- Chill a box of pipette tips at -4oC and put your reaction tubes on ice.
- Obtain DH5α cells from the –80oC freezer and thaw in palm. Store on ice for 10 minutes.
- Use a chilled tip to transfer 100 ul of the DH5α cells into each tube. Set aside one tube as a control.
- Pipet up to 5uL of the plasmid (up to 50ng per 100ul of competent cells, plasmid amount. Should not exceed 5% that of the competent cells) into each tube. Swirl.
- Store on ice for 30 minutes.
- Place tubes in incubator set to 42oC for EXACTLY 90 seconds.
- Return the tubes to ice for 2 minutes.
- Add 800uL of LB to each tube.
- Incubate at 37oC for 45 minutes, ensure that the tube has at least 2ml capacity, to properly aerate the cells.
- Spread cells on plate with appropriate antibiotic: plate between 50- 300ul each. Let dry.
- Place plates inverted in incubator at 37oC for 12-16 hours.
