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Team:Glendale CC AZ/Protocols/Transformation
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/GrowthCurve">Growth Curve Assay</a></p> | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/GrowthCurve">Growth Curve Assay</a></p> | ||
<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/NaCl">NaCl Growth Curve Assay</a></p> | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/NaCl">NaCl Growth Curve Assay</a></p> | ||
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<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Ligation">Ligation</a></p> | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Ligation">Ligation</a></p> | ||
<a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Transformation">Transformation</a></p> | <a href="https://2013.igem.org/Team:Glendale_CC_AZ/Protocols/Transformation">Transformation</a></p> | ||
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6. Incubate the cells on ice for 5 minutes. | 6. Incubate the cells on ice for 5 minutes. | ||
| - | 7. Add 200 | + | 7. Add 200 µL of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation. |
8. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. | 8. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. | ||
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12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep. | 12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep. | ||
| - | 13. Count the colonies on the 20 | + | 13. Count the colonies on the 20 µL control plate and calculate your competent cell efficiency. |
Revision as of 03:04, 23 September 2013
Glendale Community College Arizona
Protocols
Growth Curve Assay
NaCl Growth Curve Assay
Survival Growth Assay
Alkaline Lysis Plasmid Miniprep
Restriction Digest
DNA Isolation
Bioinformatics
Ligation
Transformation
Transformation Protocol
Protocol from: http://parts.igem.org/Help:Protocols/Transformation
Materials
-Resuspended DNA -Competent cells (50ul per transformation) -Ice -2ml tube (1 per a transformation) -42ºC water bath -SOC media -Petri dishes with LB agar and appropriate antibiotic (2 per transformation) -Spreader -37ºC incubator -10pg/µl RFP Control (pSB1A3 w/ BBa_J04450)
Procedure
1. Start thawing the competent cells on ice. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
2. Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
3. Add 1 µL of the RFP Control to your control transformation.
4. Close tubes and incubate the cells on ice for 30 minutes.
5. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
6. Incubate the cells on ice for 5 minutes.
7. Add 200 µL of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation.
8. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking.
9. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
10. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
11. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up.
12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
13. Count the colonies on the 20 µL control plate and calculate your competent cell efficiency.
