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Transformation
From 2010.igem.org
(Difference between revisions)
(New page: #Thaw cells on ice. #Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough). #*Note: If you are adding small volumes (~1μl), be careful to mix the culture well. ...) |
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#Let sit for 5 minutes on ice. | #Let sit for 5 minutes on ice. | ||
#*Note: For better efficiency do at least 30 minutes | #*Note: For better efficiency do at least 30 minutes | ||
| - | #Incubate cells for 15 seconds at | + | #Incubate cells for 15 seconds at 42C. |
#Incubate cells on ice for 2 min. | #Incubate cells on ice for 2 min. | ||
#Add 200 mL [[LB]] ([[2XYT]] and SOC are also suitable for greater efficiency) | #Add 200 mL [[LB]] ([[2XYT]] and SOC are also suitable for greater efficiency) | ||
| - | #Incubate for 1 hour at | + | #Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes) |
#Spread 100-300 μl onto a plate made with appropriate antibiotic. | #Spread 100-300 μl onto a plate made with appropriate antibiotic. | ||
| - | #Grow overnight at | + | #Grow overnight at 37C. |
#Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants. | #Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants. | ||
Adapted from the openwetware wiki | Adapted from the openwetware wiki | ||
Revision as of 17:48, 3 June 2010
- Thaw cells on ice.
- Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
- Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
- Let sit for 5 minutes on ice.
- Note: For better efficiency do at least 30 minutes
- Incubate cells for 15 seconds at 42C.
- Incubate cells on ice for 2 min.
- Add 200 mL LB (2XYT and SOC are also suitable for greater efficiency)
- Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes)
- Spread 100-300 μl onto a plate made with appropriate antibiotic.
- Grow overnight at 37C.
- Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
Adapted from the openwetware wiki
