8 replies to this topic

[s_laub]

The subject says it all. I need to add X-gal/IPTG to a plate, but my transformants are already on there and have grown to visible colonies.

I was thinking of adding the appropriate amount, swirling it to let it cover all the colonies, and then let it sit at 37 for a few hours.

What do you think?

[fishdoc]

s_laub, on Sep 22 2009, 10:13 PM, said:

The subject says it all. I need to add X-gal/IPTG to a plate, but my transformants are already on there and have grown to visible colonies.

I was thinking of adding the appropriate amount, swirling it to let it cover all the colonies, and then let it sit at 37 for a few hours.

What do you think?

Sounds like a bad idea, but I have no personal experience doing that. Try colony PCR on a couple dozen colonies. If it's a high copy number plasmid, you can try toothpick lysis and run it next to empty vector to look for a shift due to the insert.

[Warren]

s_laub, on Sep 22 2009, 11:13 PM, said:

The subject says it all. I need to add X-gal/IPTG to a plate, but my transformants are already on there and have grown to visible colonies.

I was thinking of adding the appropriate amount, swirling it to let it cover all the colonies, and then let it sit at 37 for a few hours.

What do you think?

I think you'll kill your bacteria.  The x-gal is in dimethyl-formamide, and bathing the cells in a nice solution of that can't be good   If i HAD to find a way to save it, I would be more tempted to swirl some LB over them and replate a small bit of the broth on a new x-gal/iptg plate, or replica plate.  Also, you can proceed without this step if your background of vector only is not expected to be too high.  Warren..

[Qundo12]

Just pick some colonies to the new X-gal/IPTG plate. If you afraid of low efficiency in ligation, just increase the number of colonies. Anyhow, the screening step of colony PCR or RE digestion is still needed, so if I were you, I would perform the colony PCR (maybe with larger number of colonies).

[s_laub]

Sorry, I should have mentioned that this is a control plate and all I'm interested in is the ratio of blue to white.

[perneseblue]

bad idea. Swirling around the X-gal/IPTG solution will smear your colonies. Cells from individual colonies will mix and you will get a mixed up mess.

Aside from picking colonies and streaking them to a plate which does contain Xgal/IPTG/Antibiotics you can plate replicate your colonies (provided that the colony density is low and individual colonies are well spaced apart). What you need is a two sheets of circular 3M whatman paper, a round block the shape of a petri dish. You can then replicate the colonies on the whatman paper by pressing the plate on to it. Remove the old plate and then press on your new Xgal/IPTG/Antibiotics plate.

That should work.

Alternatively you try overlaying the whatman paper directly onto the original plate. Get a faint imprint of the colonies. And then place the whatman paper on your new plates and then let the colonies regrow.

[almost a doctor]

perneseblue, on Sep 23 2009, 08:36 AM, said:

bad idea. Swirling around the X-gal/IPTG solution will smear your colonies. Cells from individual colonies will mix and you will get a mixed up mess.

Aside from picking colonies and streaking them to a plate which does contain Xgal/IPTG/Antibiotics you can plate replicate your colonies (provided that the colony density is low and individual colonies are well spaced apart). What you need is a two sheets of circular 3M whatman paper, a round block the shape of a petri dish. You can then replicate the colonies on the whatman paper by pressing the plate on to it. Remove the old plate and then press on your new Xgal/IPTG/Antibiotics plate.

That should work.

Alternatively you try overlaying the whatman paper directly onto the original plate. Get a faint imprint of the colonies. And then place the whatman paper on your new plates and then let the colonies regrow.

Hey s_laub, I agree that  swirling your colonies in x-gal/IPTG is a bad idea, but you can spray over instead. We used to that and it works quite nicely.   Just prepare the X-gal/IPTG at the appropriate concentration, and spray it on top of the colonies using a "perfume bottle type difusor" (sorry I really don't know how to call it, I hope this makes sense   ). Put your plates back in the incubator for 1-2h, and you should have white-blue colonies.

Hope this helps.

[MaggieRoara]

almost a doctor, on Sep 23 2009, 12:12 AM, said:

perneseblue, on Sep 23 2009, 08:36 AM, said:

bad idea. Swirling around the X-gal/IPTG solution will smear your colonies. Cells from individual colonies will mix and you will get a mixed up mess.

Aside from picking colonies and streaking them to a plate which does contain Xgal/IPTG/Antibiotics you can plate replicate your colonies (provided that the colony density is low and individual colonies are well spaced apart). What you need is a two sheets of circular 3M whatman paper, a round block the shape of a petri dish. You can then replicate the colonies on the whatman paper by pressing the plate on to it. Remove the old plate and then press on your new Xgal/IPTG/Antibiotics plate.

That should work.

Alternatively you try overlaying the whatman paper directly onto the original plate. Get a faint imprint of the colonies. And then place the whatman paper on your new plates and then let the colonies regrow.

Hey s_laub, I agree that  swirling your colonies in x-gal/IPTG is a bad idea, but you can spray over instead. We used to that and it works quite nicely.   Just prepare the X-gal/IPTG at the appropriate concentration, and spray it on top of the colonies using a "perfume bottle type difusor" (sorry I really don't know how to call it, I hope this makes sense   ). Put your plates back in the incubator for 1-2h, and you should have white-blue colonies.

Hope this helps.

How do you prepare the X-gal/IPTG solution? whats the recipe?

[Chimp]

I made the same mistake. Carefully adding ~300 microliters of IPTG/X-gal in the appropriate conc worked perfectly. If the colonies are well spaced, it is easy to inspect by eye whether they are smearing. In my case, they didn´t!