Lidstrom:Competent Cell Preparation: Difference between revisions
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**Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest. | **Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest. | ||
*You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC. | *You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC. | ||
==Electrocompetent Cells== | |||
*You can make your own electro-competent cells for electroporation. | |||
**From Amada's past mentor in undergrad: | |||
***Grow the cells overnight | |||
***Inoculate from this the next morning: generally use 200 uL/50 mL | |||
***Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice). | |||
***Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C. | |||
****Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells| this page]]. Would allow you to calculate efficiency. |
Revision as of 17:56, 2 July 2012
Back to Protocols
Chemically Competent E. Coli
Notes:
- You need fresh cells.
- Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.
- Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.
- You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.
Electrocompetent Cells
- You can make your own electro-competent cells for electroporation.
- From Amada's past mentor in undergrad:
- Grow the cells overnight
- Inoculate from this the next morning: generally use 200 uL/50 mL
- Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).
- Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
- Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page. Would allow you to calculate efficiency.
- From Amada's past mentor in undergrad: