Whaaaaa my cells didn't grow:(

Xira

So I wake up at 5 frikkin AM so I can make some cells electro competant for a knockout and what do I find when I get to the lab at 6 !@##$!@ing am? Seems Salmonella Thypi dosen't like the pkd46 plasmid....Or mabey it wasn't selected for or mabey the plates weren't Amp+ or mabey it's got a restriction enzyme that's chewing it up or something. Argh. Annoying.

Serien

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Originally posted by Xira 
Seems Salmonella Thypi dosen't like the pkd46 plasmid

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aw c'mon, we all know that

lol..just kidding

are you majoring in some sort of science?

or are you in that field?

i hope you are....instead of just making plasmaidoc +lierbadjvielad = hypoglocymeahsodflfghje!

BrotherAlpha

Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).

Then again, you might just be unlucky.  Try again tomorrow!

Dewfire

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Originally posted by BrotherAlpha

Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).
Then again, you might just be unlucky.  Try again tomorrow!

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Yea thats what I always try too...

BigPeeler

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Originally posted by BrotherAlpha

Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).
Then again, you might just be unlucky.  Try again tomorrow!

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... my thoughts exactly 

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Xira

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Originally posted by BrotherAlpha

Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).
Then again, you might just be unlucky.  Try again tomorrow!

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I think it's dat last one. Or mabey it was grown at a plasmic-destroying temprature(temp sensitive replicon).

I didn't do the transfection myself...Suffice to say that if _I_ had done it it would have worked:) Useing electroporation. The postdoc in charge of the lab said she'd take care of it and proceeded to make the transformants useing the "good enough(tm)" Ca-CL+ method....This produced transformants that grew on what (We think) was an Amp plate. Just that when put into an Amp+ culture to prepare them for further electroporation they all died. As for what's wrong...Eh...Well perhaps the plate wasn't really Amp, or perhaps the plasmid she was useing didn't actually have plasmid in it.

After some investigation it SHOULD work(Nothing in Salmonella prohibits the Red-46 gene disruption protocol) and it's even been done successfully in other strains...As to what's wrong no clue.

MegaWolf

Have you tried transconducting the amp plate? It may change the protolithocell number to 4.98 instead of 5.12. That should stablize the co-refronsire making the operation not only safer but more successful.

Xira

*throws a sallabaloon at Megawolf*

BrotherAlpha

Lol! Sounds like there's too many "if's" in that procedure.  If's aren't good in transfection