Protocol/1

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Revision as of 00:59, 26 October 2010

TEAM ALBERTA

Protocol 1: Transformations

Regeants:

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 -4 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.
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 42^OC for EXACTLY 90 seconds.
Return the tubes to ice for 2 minutes.
Add 800uL of LB to each tube.
Incubate at 37^oC 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 37^oC for 12-16 hours.

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@@ Line 14: / Line 14: @@
-What you will need:
+Regeants:
-* Competent DH5α cells (50 uL per transformation)
+* Competent DH5α cells (100uL per transformation)
-* 2 uL of plasmid you want to transform
+* up to 5uL of plasmid you want to transform
-* 800 uL of LB (non-contaminated) per transformation
+* 800uL of LB (non-contaminated) per transformation
 * Plate with correct antibiotics
@@ Line 25: / Line 25: @@
 * Chill a box of pipette tips at -4 C and put your reaction tubes on ice.
-* Obtain DH5α cells from the –80 C freezer and thaw in palm. Just as they thaw, store on ice for 10 minutes.
+* Obtain DH5α cells from the –80 C freezer and thaw in palm. Store on ice for 10 minutes.
-* Use a chilled tip to transfer 50 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 2uL of the plasmid (up to 25ng per 50 ul of competent cells, plasmid amt. 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.
-* Place tubes in incubator set to 42 C for EXACTLY 90s.
+* Place tubes in incubator set to 42<sup>O</sup>C for EXACTLY 90 seconds.
-* Return the tubes to ice for 2 min.
+* Return the tubes to ice for 2 minutes.
 * Add 800uL of LB to each tube.
-* Incubate at 37 C for 45 min, ensure that the tube has at least 2ml capacity, to properly aerate the cells.
+* Incubate at 37<sup>o</sup>C 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 37 C for 12-16 hours.
+* Place plates inverted in incubator at 37<sup>o</sup>C for 12-16 hours.
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