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Team:Hawaii/Test Competent E. Coli
From 2008.igem.org
Contents |
Test Competent E. coli DH5a
- Test our competent cells against Callahan competent cells
Methods
Materials
- Transported 50uL of competent cells from Callahan Lab on ice
- 100uL batch 1 competent cells made at OD600 ~0.6 (split into two 50 uL aliquots 4-18a,4-18b)
- 100uL batch 2 competent cells made at OD600 ~0.3 (split into two 50 uL aliquots 2-17a,2-17b)
Procedure
- Thaw plasmid on ice
- Add 1uL plasmid pBluescript
- Incubate plasmid+competent cells on ice for 30 minutes
- Heat shock 60 sec at 42C
- Add 250 μl SOC
- Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
- For plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, growing for 2 hours yields many more colonies
- Ampicillin and kanamycin resistant transformants appear to do fine with 1 hour growth
- Plate 20 uL dilutions 1/1, 1/10, 1/100 to test competency
Results
Discussion
Next time, remember to aliquot out and transform only 50 ul of the competent cells.
- What was learned and how to do future experiments differently.
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
