IGEM:MIT/2006/Notebook/2006-6-10
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Smell enzyme transformation
There were very few colonies:(
| Enzyme | BL21(DE3) | DH5α |
|---|---|---|
| pUC19 ctrl | 0 | 0 |
| SAMT | 7 | 0 |
| BAMT | 2 | 0 |
| BSMT | 0 | 0 |
Debugging the transformation
- Were the pUC19 vector transformations plated on the Amp plates? In future, if the plates aren't already labeled, they should be labeled as soon as they come out of the cold room.
- Might the vector have had a different antibiotic resistance than Amp?
- You added 2μl of the pUC19 vectors to the cell mix? Did we correctly calculate how much of this we should add.
- Did we correctly calculate the concentration of the reconstituted DNA from the filter paper?
- We omitted the 42C heatshock since it seems unnecessary for TSS competent cells. Maybe the heat shock is necessary for the BL21(DE3) one shot chemically competent cells from invitrogen?
- RS 13:41, 10 June 2006 (EDT): Yes, the Invitrogen protocols generally call for heat shock.
- Did we let the spreader cool down after flaming before touching the cells?
- For the transformations into BL21(DE3), maybe we should add glucose to the plates and the 2xYT medium to ensure that expression of the T7 RNAP is well shut off?
- I (BC) am going to plate more of the transformed cells.
- To help the debugging, its important that you record all the details of the protocol you used. Its great that you linked to the transformation protocol you used on yesterday's notebook page, but you should remember to add any alterations to the protocol, for examply, we chose not to do the heat shock step, we used 2xYT media for the 37C incubation, you plated 200ul of cells etc.
