12 replies to this topic

[KatieB]

Hi,

I'm having repeated problems with some clones of bacterial and cytokine vectors.  I made most of these a year ago (all fine) and froze down copies of them in glycerol stocks (never a problem before).  when bringing them up fresh i get colonies (approx 20) but when i lyse and pcr them i get either no product, or a faint product which when miniprepped (Qiagen) and tested on realtime PCR has nothing!  I have done PCR of these minipreps and get no product.  also tried new miniprep kit, fresh broths and agars, lysis buffer etc.  I still have a few things I can try, and am going to send one of these minipreps for sequencing, but wondered if anyone had had similar problems for fast solution!  
I have been told it could be that my plasmid has fallen out of vector, however I have the same problem with a number of different clones and dont think the same problem would have happened in all of them!

Thank you for your attention,

KT

[arush]

I dont see why u had to preserve plasmids with glycerol. That could be a reason for the very low transformation efficiency. (i know this from someone's experience who had unknowingly resuspended plasmids in 50% glycerol and never got any colonies again) . And in case it is thae cultures that u have preserved in glycerol...fresh transformation of plasmid should work.
Good luck

[rkay447]

When you say you froze down copies in glycerol stocks, you mean you made glycerol stocks of the bacteria, right?  And then you restreak the bacterial glycerol stock, right?  Anyway, my glycerol stocks never yeild much, if any, DNA.  I'll get colonies, expand and prep and feel lucky if I can get up to 50 ng/ul.  Not sure why or what I'm doing differently since everyone else in the lab uses these stocks but I always have to retransform the DNA in order to get good DNA preps.  Now, rather than glycerol stocks, I have a box of "stock DNA" with 20ul of each clone.  When I need to amplify I just retransform 0.5ul.  See if you can amplify with a fresh transformation.  If not, then you have a serious problem.

[cellcounter]

KatieB, on Feb 6 2009, 04:46 AM, said:

Hi,

I'm having repeated problems with some clones of bacterial and cytokine vectors.  I made most of these a year ago (all fine) and froze down copies of them in glycerol stocks (never a problem before).  when bringing them up fresh i get colonies (approx 20) but when i lyse and pcr them i get either no product, or a faint product which when miniprepped (Qiagen) and tested on realtime PCR has nothing!  I have done PCR of these minipreps and get no product.  also tried new miniprep kit, fresh broths and agars, lysis buffer etc.  I still have a few things I can try, and am going to send one of these minipreps for sequencing, but wondered if anyone had had similar problems for fast solution!  
I have been told it could be that my plasmid has fallen out of vector, however I have the same problem with a number of different clones and dont think the same problem would have happened in all of them!

Thank you for your attention,

KT

This generally does not happen with bacterial glycerol stocks, even if handled badly, but you may want to think of the following:

1. That you are using the right antibiotics (sometimes you forget that it was kan resistant, not ampR)

2. make sure the freeze had not broke down for extended period during last one year.

3. Plasmids don't fall off, but if at the time of freezing down, if you had cultured them for a long time in absence of antibiotic, they may have falled down.

4. Check your current reagents. Which you already did!

5. As there is a problem, I suggest streaking the cells first on Ab free plates or even growing in liquid media for an hour so without Ab. In case if the resistance gene dose has gone down.

[scolix]

It is possible for some glycerol stocks to go bad (no colonies after streaking), but it very very rare. Ofcourse, one suspects that the protocol for these glycerol stocks might have had some error or in preparation of the stocks. I hope you have taken these into account.

Recheck all solutions, and try to directly grow a liquid culture (without antibiotics) from the glycerol stocks.

[KatieB]

Hi
Thank you for all these comments.  I have used this same glycerol stock method for years (yes i mean inoculation of a freezer vial with broth and 10%glycerol) and not had any problems, and my freezer has been held at constant -80 the whole time.  
I have also made fresh clones which have exactly the same problem, so it can not be my glycerol stocks it must be something in media?  I have made fresh ampicillin (this is the right antibiotic for pGEM T-easy vectors).

rkay447 - you said instead you keep a DNA stock box - is this just pure DNA, PCR products or DNA in a vector (can this be kept longterm?)?  I use purified amplified bacterial DNA for my cloning, and have always thought that purified PCR products should not be kept for use for more than couple of weeks?  

I will try growing without ampicillin to see if it makes a difference.  Am also just going to throw everything out AGAIN and start fresh again! always a good method when something randomly goes wrong in the lab I find!   i'll keep you posted!

Thanks again,

Katie

[perneseblue]

KatieB, on Feb 9 2009, 06:49 AM, said:

is this just pure DNA, PCR products or DNA in a vector (can this be kept longterm?)?  I use purified amplified bacterial DNA for my cloning, and have always thought that purified PCR products should not be kept for use for more than couple of weeks?

If the plasmid is prepared well it can be kept for over a decade.
PCR products and DNA fragments can be kept in a form usable for DNA ligation for over 2 years at -20 C. There is some lost in efficiency but not much.

There are two way one can keep a plasmid.

1 - keeping an e coli strain containing the plasmid
2 - keeping the plasmid as pure DNA.

I prefer keeping my plasmids as DNA. A large quantity of DNA can be kept, so the probability of losing the plasmid (even in situations of power outage and repeated freeze thaw cycles) is small.

[cellcounter]

You can also spot the plasmids on 3M paper, put it in a plastic sheet, and keep a notebook that way. Our neighbour lab supervisor has an album of such plasmids.

She has made multiple marked spots on each 3M sheet, and whenever someone requests plasmids, she just cuts a sqaure and sends it. She has been keeping it this way for over a decade now.

[perneseblue]

cellcounter, on Feb 9 2009, 01:00 PM, said:

You can also spot the plasmids on 3M paper, put it in a plastic sheet, and keep a notebook that way. Our neighbour lab supervisor has an album of such plasmids.

She has made multiple marked spots on each 3M sheet, and whenever someone requests plasmids, she just cuts a sqaure and sends it. She has been keeping it this way for over a decade now.

that is interesting.

[Qundo12]

cellcounter, on Feb 10 2009, 06:00 AM, said:

You can also spot the plasmids on 3M paper, put it in a plastic sheet, and keep a notebook that way. Our neighbour lab supervisor has an album of such plasmids.

She has made multiple marked spots on each 3M sheet, and whenever someone requests plasmids, she just cuts a sqaure and sends it. She has been keeping it this way for over a decade now.

Yeah, so creative, plasmids can be easily stored at RT.
@KatieB: I have a reference for you. This sometimes happens in some labs and I also got this problem before, a little bit less serious, just the plasmid yield was very low. In this paper, they grew the cell in the media containing the excess concentration of ampicillin, since this would select the cells containing high number of plasmid copy. Hope this helps   .

Attached Files

•  Detection_of_lost_plasmids_from_E._coli_using_excess_ampicilin.pdf   71.45K   221 downloads

[KatieB]

Quasimondo, on Feb 10 2009, 02:04 AM, said:

cellcounter, on Feb 10 2009, 06:00 AM, said:

You can also spot the plasmids on 3M paper, put it in a plastic sheet, and keep a notebook that way. Our neighbour lab supervisor has an album of such plasmids.

She has made multiple marked spots on each 3M sheet, and whenever someone requests plasmids, she just cuts a sqaure and sends it. She has been keeping it this way for over a decade now.

Yeah, so creative, plasmids can be easily stored at RT.
@KatieB: I have a reference for you. This sometimes happens in some labs and I also got this problem before, a little bit less serious, just the plasmid yield was very low. In this paper, they grew the cell in the media containing the excess concentration of ampicillin, since this would select the cells containing high number of plasmid copy. Hope this helps   .

That is really interesting! thank you.

[KatieB]

Hi everyone, thank you for all your comments and help.  I have managed to get everything working again, I made fresh ampicillin and all fresh media and still had afew problems with making fresh clones. so went back and got a fresh vector kit (my other one was quite new),. after doing this my cloning is working again and i've now brought up stocks from freezer of old plasmids which are all growing fine and working perfectly on realtime PCR   Therefore, basically I must have had a multitude of problems with both media and vector kit.  advice - never rely on expiry dates on kits!!!  

Anyway, thanks for everything,

Katie

[Qundo12]

KatieB, on Feb 22 2009, 03:23 AM, said:

Hi everyone, thank you for all your comments and help.  I have managed to get everything working again, I made fresh ampicillin and all fresh media and still had afew problems with making fresh clones. so went back and got a fresh vector kit (my other one was quite new),. after doing this my cloning is working again and i've now brought up stocks from freezer of old plasmids which are all growing fine and working perfectly on realtime PCR   Therefore, basically I must have had a multitude of problems with both media and vector kit.  advice - never rely on expiry dates on kits!!!  

Anyway, thanks for everything,

Katie

Congratulation, nice to hear that ^^. I also agree that "never rely on expiry dates on kits" because recently I found out one of my Kit was overdued for 6 months but thank God, it still worked well, lol