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Team:Amsterdam/Notebook/Protocols/Transforming Competent Cells
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==Transforming Competent Cells== | ==Transforming Competent Cells== | ||
'''Estimated time: 3 hours''' (plus 12-14 hour incubation)<br> | '''Estimated time: 3 hours''' (plus 12-14 hour incubation)<br> | ||
| - | + | This protocol is used for transforming the ligated plasmids or resuspended DNA into [https://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Making_Competent_Cells self-made competent cells]. | |
| - | + | ||
| - | + | ||
===Materials needed=== | ===Materials needed=== | ||
| - | *Resuspended DNA (''Resuspend well in | + | * Ligated plasmid or Resuspended DNA (''Resuspend well in 10 µl dH20, pipette up and down several times, let sit for a few minutes'') |
| - | *Competent cells ('' | + | *Competent cells (''50 µl per a transformation'') |
*Ice | *Ice | ||
| - | * | + | *42ºC water bath |
| - | * | + | *37ºC incubator |
| - | *SOC (''check for contamination!'') | + | *SOC (<b>''check for contamination!''</b>) |
| - | *Petri dishes with LB agar and appropriate antibiotic | + | *Petri dishes with LB agar and appropriate antibiotic |
===Protocol=== | ===Protocol=== | ||
# Start thawing the competent cells on crushed ice. | # Start thawing the competent cells on crushed ice. | ||
| - | # Add 50 µL of thawed competent cells and then 1 - 2 | + | # Add 50 µL of thawed competent cells and then 1 - 2 µl of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice. |
# Incubate the cells on ice for 30 minutes. | # Incubate the cells on ice for 30 minutes. | ||
# Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells. | # Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells. | ||
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# Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated) | # Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated) | ||
# Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. '''Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.''' | # Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. '''Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.''' | ||
| - | # Label | + | # Label a petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 200 µl of the transformation onto the dish, and spread. This helps ensure that you will be able to pick out a single colony. |
# Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria. | # Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria. | ||
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Revision as of 10:21, 19 September 2011
Transforming Competent Cells
Estimated time: 3 hours (plus 12-14 hour incubation)
This protocol is used for transforming the ligated plasmids or resuspended DNA into self-made competent cells.
Materials needed
- Ligated plasmid or Resuspended DNA (Resuspend well in 10 µl dH20, pipette up and down several times, let sit for a few minutes)
- Competent cells (50 µl per a transformation)
- Ice
- 42ºC water bath
- 37ºC incubator
- SOC (check for contamination!)
- Petri dishes with LB agar and appropriate antibiotic
Protocol
- Start thawing the competent cells on crushed ice.
- Add 50 µL of thawed competent cells and then 1 - 2 µl of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.
- Incubate the cells on ice for 30 minutes.
- Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
- Incubate the cells on ice for 5 minutes.
- Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)
- Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
- Label a petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 200 µl of the transformation onto the dish, and spread. This helps ensure that you will be able to pick out a single colony.
- Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.
