Deepa Galaiya wrote: + control DNA sent by Promega was successfully transformed. None of the iGEM DNA was though. This is a problem. We can just try to grow up some glycerol stocks or repeat the transformation for next week's lab.
John Cumbers wrote: mmm, could it be that the cells got warm and were no longer competent perhaps? Can you think of what else it might be? Are there some other competent cells left over from the summer still, could somebody repeat it with those, and maybe get some from Jamie Gagnon as another control? Or is there a better construct a reliable part that could be transformed (e.g maybe our plate is registry plate is bad or maybe the parts you chose were bad?) let me know what you think as we really want to get on top of it now I think and get them working in the future.. cheers, John
Jeffrey Hofmann wrote: Deepa, any idea how much DNA was used of pUC19 relative to the iGEM plasmids? Also, I suppose that maybe the iGEM DNA went bad somehow, but I was under the impression that it would last a long time since it's lyophilized. Are we sure that all of the plasmids used were Amp resistant, and are we sure that the plates had Amp and no other antibiotics? Also, did you do the iGEM part that I didn't have time to finish, and that didn't work either? that would certainly indicate that something either the iGEM DNA is bad, or we used different amounts of iGEM DNA and pUC19. Approximately how many colonies did you get on the pUC19 plate?
Jeffrey Hofmann wrote: Okay, it's probably the concentration. Let me know when you find out what it is. However, this is still a problem because we can't change the concentration of the iGEM DNA. We may want to look into getting a different transformation protocol, because these should be more efficient. Maybe we should try again and add back in the 90' at 37 C step to see if that makes a difference.
Could you guys describe what you did and include your protocol? I'm a little confused as to what happened from the posts on this discussion so far. So if you could clarify a bit more, I can try to help you out!
That's not too different from what we do but why don't I give you our protocol to try if you can. The one major difference is that we do our recovery in SOC (SOB plus glucose).
- Resuspend DNA in 10ul of diH2O
- Add 1ul of resuspended DNA to 50ul thawed competent cells (on ice) (we make our own competent cells: http://openwetware.org/wiki/TOP10_chemically_competent_cells)
- Incubate on ice for 30 minutes
- Heat shock in 42 degrees water bath for 1 minute
- Incubate on ice for 5 minutes
- Take off ice, add 200ul of SOC (http://openwetware.org/wiki/SOC)
- Recover in 37 degree incubator for 2 hours
- Plate all 250ul on appropriate antibiotic agar plate and incubate overnight at 37 degrees
Also, how did you guys store your kit plates? The DNA in the plates is not lyophilized (freeze-dried). It is just dried under the hood. In any case, that shouldn't make a difference. Which parts were you trying to transform?
We should put together a consensus protocol explaining how to use the DNA the registry ships out.
We could be begin by reconciling the minor differences in the three protocols already listed: John's, Meagan's, and the one at Help:IGEM 07 Parts Kit.
Or perhaps it should be a set of smaller protocol "modules", like 0. Resuspension 1.A Transformation (Chemical) 1.B Transformation (Electroporation) 2. Incubation, growth 3.A Glycerol stocks 3.B miniprep
Cool, we're planning to repeat it this week, we'll try the SOC as you suggest. It's been in the -20 since June. I agree consensus protocols would be good, video protocols even better. let's see what we can do. cheer,s John