Joyce:Transformation: Difference between revisions
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(New page: =Transformation= ==Procedure== #Obtain an ependorff of competent cells #Place the cells on ice and let them thaw on ice (about 10-15 minutes) #Gently stir the cells with a pipette tip to...) |
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=Transformation= | =Transformation= | ||
==Procedure== | ==Procedure (for E. coli XL2-Blue)== | ||
#Obtain an ependorff of competent cells | #Obtain an ependorff of competent cells | ||
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#Discard 300 ul of the supernatant | #Discard 300 ul of the supernatant | ||
#Resuspend the pellet in the remaining 150 ul | #Resuspend the pellet in the remaining 150 ul | ||
#Plate | #Plate and incubate at 37*C overnight |
Revision as of 10:14, 1 September 2009
Transformation
Procedure (for E. coli XL2-Blue)
- Obtain an ependorff of competent cells
- Place the cells on ice and let them thaw on ice (about 10-15 minutes)
- Gently stir the cells with a pipette tip to resuspend them
- Pipette 50 ul of cells into an ependorff
- With gentle stirring, pippette 5 ul of a 20 ul ligation reaction into the ependorff
- Place the ependorff on ice for at least 30 minutes
- Prepare the heating block for 42*C
- Heat shock the transformation mixture for 45 seconds at 42*C
- Put the transformation mixture on ice for 2 minutes
- Add 400 ul of sterile YT to the transformation mixture
- Mix gently by inverting several times
- Incubate at 37*C for at least 45 minutes
- Centrifuge at full speed for 2 minutes
- Discard 300 ul of the supernatant
- Resuspend the pellet in the remaining 150 ul
- Plate and incubate at 37*C overnight