Disappearing colonies? What happened? Help! - (Dec/17/2012 )

Last week I tried to transform some E Coli from a TA Cloning kit that I've always gotten to work for me. They were old plasmid that someone had already made a long time ago that were in the freezer. The concentration was low...10^6 molecules per microliter. When I first tried to transform them, I only added 1 uL of plasmid to 25uL of cells and got no colonies. Then after I talked to the post doc who originally made these plasmids, his advice was to bump up everything so I used 10uL of plasmid in 50uL of cells and got these fat clear colonies, rather than small tight white ones like I expected. I figured I had just let them grow out too long, but it was Friday and I didn't want to come in on the weekend so I put them in the fridge until today and I had planned on streaking one of the colonies on a plate to purify/separate them out overnight to get the tiny white colonies before setting up culture tubes for minipreps tomorrow. I went to look at my plates this morning, and they are blank. The "fat clear colonies" are gone and the plates are empty. I am totally confused. Has anyone seen this happen before?

Sounds like the plasmid somehow conferred transient antibiotic resistance, and then the either the plasmid has disappeared or the resistance has gone. I you will have to repeat the transformation anyway. I would recommend that you not add more DNA than 5% of the cell volume - too much can inhibit the transformation process.

If the DNA is from a midi or mini style prep, then you may want to check the integrity of the DNA before transforming, too many freeze/thaw cycles and the DNA will be degraded significantly.

Okay, well here's my confusion about your reply.

the 5% recommendation - is that by volume or by mass of DNA to mass of cells or number of molecules to number of cells or what?

I had 10^6 molecules to 25 uL of cells the first time and 10^7 molecules to 50uL the second time I tried it. (Kit; StrataClone PCR Cloning Kit, Catalog #240205)
Manual: http://www.chem-agilent.com/pdf/strata/240205.pdf - I can't find in this manual what the cell number density is in 1uL of the mixture, nor any instructions for re-transforming with already-generated plasmid. (only instructions for how much PCR product to add to the empty vector.)

The statement "too many freeze/thaw cycles can degrade DNA" has been told to me over and over, but what never gets explained is how many "too many" is, and also I've recieved conflicting information about this fact anyway. That, yes, it CAN degrade DNA over time but plasmid are pretty resilient compared to PCR products and genomic DNA when it comes to freeze/thawing, and also, if these things are stored frozen and the researcher who I got them from is used to storing them at -20 for a year and being able to bring them back out and re-transform them no problem, then these stocks should be of fine enough quality.

Oh, also, i replied too fast before and I meant to say that when I checked the concentration of my plasmid on our Nanovue, it was 0.3ng/uL. So, not a lot at all. That would mean that 10uL would be 3ng/uL of plasmid. the kit recommends between 5ng-50ng of PCR product to ligate to vector, so I'm not sure how closely that relates to the amount of whole plasmid to add to the cells. Like I said above, the kit offered no instructions for how to transform with a plasmid that is already made.

So if it's 5% ng/ng then I added 3ng/? ng for cells

if it's volume/volume, then I added too little the first time (1uL : 25uL ) and way too much (10ul : 50uL )the second time

if it's molecule to cell: then ????

So basically I need to develop a more detailed understanding of this but I don't know where to begin to look since the kit instructions don't include much of this.


Edit....

One more thought. I sometimes quickly brush the flame over the top of my plates to pop air bubbles after pouring. This is something my boss told me to do, but she has never watched me do it before. I am wondering if doing that ends up destroying the antibiotics on the surface of the plate and if so, would it be acceptable to make up antibiotics at the appropriate concentration (dilute to 50ug/mL) in a volume of about 50uL and then spread it on the plate and let it soak into the plate before I plate the bacteria? Would that run the risk of adding too much antibiotic then just in case the flame has no effect on the antibiotic?

betamarie on Mon Dec 17 22:20:56 2012 said:

by volume.

This is true, too many is too many, sometimes it may be as few as 1, sometimes 20 or more, it depends on a range of factors including speed of freeze and thaw, the concentration, solute, etc. If someone else has brought them up before and transformed, then can't you use their glycerol stock?

betamarie on Mon Dec 17 22:45:41 2012 said:

The transformation should be the easy step. Do a +ve control of a plasmid such as puc that you know will work. This will tell you if there is something wrong with the transformation or the plates.

Should be fine. I do it all the time too and never had any problems. The antibiotic will re-diffuse back into that area anyway.

The concentration measurement is almost certainly not accurate at 3 ng/ul. I would suggest that your cells are not particularly competent, and you need very competent cells for this to work. Commercial highly chemically competent cells, or alternatively, electrocompetent cells transformed with an elecroporator. You should prepare a pUC19 standard at similar low concentration diluted into TE and use it to make sure your cells and protocol are sufficiently competent.

I'm not sure what you mean by the statement "your cells are not particularly competent and you need very competent cells for this to work." I underlined the parts that confuse me.

1. "Not particularly competent" - Are you indicating that this particular kit, the strataclone solopack, is the wrong kind of kit for this task? If so, why? Or are you indicating that these particular tubes of bacteria that I used were damaged somehow because of my technique? I don't think they were because I am careful about never letting them leave the ice until the heat shock step.
2. "This" - Do you mean that adding older, already made plasmid using some other kind of vector to cells is harder than adding freshly ligated plasmid using the vector that is designed for those cells, so you need better cells in order for it to work? Or plasmid at low concentration requires better cells?

I'm going to call the company today and find out what the maximum volume or mass of already-made plasmid this particular kit can take. We have a lot of other competent cell kits that I could try, but this is the one I have used before so I chose this one. Sorry, I'm a beginner.

betamarie on Wed Dec 19 14:35:18 2012 said:

By this Phage means that your cells may not have a high enough transformation efficiency to be able to get colonies with the very low DNA concentration. Ones from the kit should be OK, provided they have not previously been thawed. You should thaw the cells quickly and then transfer to ice.

Purely that your transformation experiments are not working currently so they may require some special conditions. Adding old plasmid shouldn't be any more difficult than using freshly prepared stuff. Low concentration requires a higher level of competency in the cells.

Don't apologise, we all have to start learning somewhere. If you have access to it, try having a read through the chapters relating to plasmids and competent cells in Sambrook et al, Molecular Cloning: a laboratory manual. Any of the editions should be a good starting point but the most recent (AFAIK) 2004 edition has a lot of additional background that will help. Especially note the references therein.

This makes sense. The person whose plasmids I am trying to transform left me with 1 million molecules per microliter of plasmid, about 40uL total. With a 5.6kb plasmid, that's not even a nanogram of plasmid. The "tried and true" DH5 alpha cells need a minimum of 1ng to get going. I'm guessing the Solopack cells i tried to use, that I mentioned above, probably require the same order of magnitude. I called Strategene tech support this morning to ask them what they recommended. They suggested I try DH5 alpha cells, but they also were not aware of my very low concentrations. I think the low concentration is the issue. For something to take up plasmid on the picogram level, I can see why I would need very chemically competent cells.

.....I can also now see why I should have raised an eyebrow at my "0.3ng/uL" reading on the nanovue. I should have known better than to trust that nanoview. It's so tempting to try to read things on it because it's so quick ... and not accurate unless you have a really concentrated sample. Shame on me for wasting all these cells when I knew better. I'll probably just go back to the beginning and do new PCRs and re-clone everything. Thanks for all your help! Sorry if I seemed testy! This is pre-holiday crunch week and I should not have tried to do something "real quick." No such thing. =)