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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= | ==Procedure (for E. coli XL2-Blue)== | ||
#Thaw cells | |||
# | |||
#Pipette 50 ul of cells into an ependorff | #Pipette 50 ul of cells into an ependorff | ||
#With gentle stirring, pippette 5 ul of a 20 ul ligation reaction into the ependorff | #With gentle stirring, pippette 5 ul of a 20 ul (about 100 ng? *****) ligation reaction into the ependorff | ||
#Place the ependorff on ice for at least 30 minutes | #Place the ependorff on ice for at least 30 minutes | ||
#Heat shock the transformation mixture for 45 seconds at 42*C | #Heat shock the transformation mixture for 45 seconds at 42*C | ||
#Put the transformation mixture on ice for 2 minutes | #Put the transformation mixture on ice for 2 minutes | ||
| Line 18: | Line 13: | ||
#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 | ||
==Notes== | |||
*You can do a cheap-o transformation to quickly check something by incubating 100 ng of DNA with cells for 30 minutes then incubate O/N at 37°C | |||
==Solutions== | |||
YT | |||
Latest revision as of 20:58, 14 September 2009
Procedure (for E. coli XL2-Blue)
- Thaw cells
- Pipette 50 ul of cells into an ependorff
- With gentle stirring, pippette 5 ul of a 20 ul (about 100 ng? *****) ligation reaction into the ependorff
- Place the ependorff on ice for at least 30 minutes
- 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
Notes
- You can do a cheap-o transformation to quickly check something by incubating 100 ng of DNA with cells for 30 minutes then incubate O/N at 37°C
Solutions
YT
