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Team:University of Chicago/Notebook/Transformations
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(New page: ==Chemocompetent cells== ===General transformation== #Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) * This is at 10 pg/_l or 10-5 _g/_l * This can be made by ...) |
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# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated | # 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. | * using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. | ||
| - | * For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol | + | * For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies |
| - | and tetracycline resistant, we find growing for 2 hours yields many more colonies | + | |
* Ampicillin and kanamycin appear to do fine with 1 hour growth | * Ampicillin and kanamycin appear to do fine with 1 hour growth | ||
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads | # Plate 20 _l on AMP plates using sterile 3.5 mm glass beads | ||
Revision as of 15:04, 8 August 2008
Chemocompetent cells
=General transformation
- Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
- This is at 10 pg/_l or 10-5 _g/_l
* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
- Hold on ice 0.5 hours
- Heat shock 60 sec at 42C
- Add 250 _l 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 our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
- Ampicillin and kanamycin appear to do fine with 1 hour growth
- Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
- Good cells should yield around 100 - 400 colonies
- Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
- We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA
