Lidstrom:Protocols: Difference between revisions
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==E. Coli== | ==E. Coli== | ||
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]] | *[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]] | ||
==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 home: Lidstrom
General Lab:
- Health & Safety
- Finding Chemicals
- Autoclave
- Sterile technique
- E. Coli basics
- Preparing Freezer Cell Stocks
- Pouring Media Plates
Cell Culture:
DNA/RNA:
DNA
- Intro: Building with DNA (in progress)
- Colony PCR
- Digestion with Restriction Enzymes
- Diluting Primers
- Gibson Assembly (In progress)
- Ligation (in progress)
- Loading Dye Recipe
- Miniprep (in progress)
- Oligo Orders (in progress)
- Overlap Extension PCR (in progress)
- PCR (in progress)
- Purifying DNA (in progress)
- Sequencing with GeneWiz
- Site-Directed Mutagenesis (in progress)
- Transformation (in progress)
RNA
E. Coli
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: