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Stock not growing. Can i re-grow Plasmid from sample?


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#1 cm13

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Posted 04 July 2010 - 04:40 AM

I recieved a gfp-tagged construct which i transformed into electrocompetent e.coli cells.
I then plated the cells on agar plates with kanamycin, to get growth of my specific construct.
I grew up one of the colonies in lb broth, and i did a plasmid purification step.
I also made glycerol stocks at the time, which i froze at -80C

That was 3 months ago.

I still have some of my purified plasmid left over, but i wanted to make more.
So i took some of the glycerol stock out of the -80C.
I took 5ul and placed it in a bacterial tube, along with 5ul of kanamycin and 5mls of LB broth.
After rotating for 8 hours, nothing had grown. After 24 hours, there was still nothing.

I tried a different approach and did streak plates of my stock.
Nothing grew after leaving it overnight. After 48 hours, i still saw nothing.


My questions are:

1) IS there something else i should try with the glycerol stock?
2) Is it possible to take the purified plasmid that i have, and repeat everything again, only this time, transforming the plasmid into ecoli cells instead of the construct? Would i go about doing this in the same way?

#2 phage434

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Posted 04 July 2010 - 05:02 AM

It sounds as if your glycerol stock is dead. For important strains, it helps to test the glycerol after -80 freezing. At this point, you probably won't get viable cells from it. Given that you have plasmid DNA it should be very easy to re-transform. For future reference, you should not throw out glycerol stocks that are dead, if they are valuable. You can run a miniprep on them to recover plasmid DNA as a last resort.

A typical way to fail with making glycerols is to omit vortexing the tube after adding glycerol and cells. There may be a spot in your tube that has viable cells, if this has happened.

#3 Adrian K

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Posted 04 July 2010 - 08:44 AM

I totally agree with phage434,

However, I share with you my last gamble choice: use all of the total glycerol stock, add into 6-8x volume of growing broth (and grow under shaking condition)...if you believe there might be still viable cells there. Try plate the whole thing after 48 hours. I know this may not be a good method, and although I had use this method for a few times to get some of my rare archive strains back, but not all the time I succeed in revive it. Still, this is something I tried before with some but not all success.
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#4 molstudent

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Posted 04 July 2010 - 10:18 AM

I have two questions so i can try to figure out your problem:

1) how did you make your glycerol stock? (how much culture vs how much glycerol)

2) when you made the glycerol stock and prepped your plasmid before, the prepped plasmid was the correct plasmid right? (you got your plasmid when you prepped the culture)

The reason i asks these are because for (1), if your glycerol stock is frozen solid in the -80C and you thawed it to take out 5ul, you may have killed your cells by thawing them. if your glycerol stock is frozen solid, the correct way is to scrape off some of the cells.

The reason i ask about question (2) is because you didn't mention a growth phase in liquid media w/o selection after transformation of your plasmid before you plated so i'm not sure if you did this. For kanamycin this is necessary or efficiency goes way way down so your colonies on your plate could be mutants that actually do not contain your plasmid.

#5 cm13

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Posted 04 July 2010 - 02:10 PM

I have two questions so i can try to figure out your problem:

1) how did you make your glycerol stock? (how much culture vs how much glycerol)

2) when you made the glycerol stock and prepped your plasmid before, the prepped plasmid was the correct plasmid right? (you got your plasmid when you prepped the culture)

The reason i asks these are because for (1), if your glycerol stock is frozen solid in the -80C and you thawed it to take out 5ul, you may have killed your cells by thawing them. if your glycerol stock is frozen solid, the correct way is to scrape off some of the cells.

The reason i ask about question (2) is because you didn't mention a growth phase in liquid media w/o selection after transformation of your plasmid before you plated so i'm not sure if you did this. For kanamycin this is necessary or efficiency goes way way down so your colonies on your plate could be mutants that actually do not contain your plasmid.



1) I went with 50ul of the cells and 50ul of Glycerol. Would this have been too much, or not enough?
2) I'm pretty sure that the plasmid i ended up getting was the correct one.

3) I didnt let the stock thaw out fully, but at the same time i did let it thaw a little instead of scraping off the cells.

4)Sorry, i forgot to add that i let the transformed cells grow in SOC broth. This is what i plated out first. I grew colonies from the plates, and took a colony and grew it up in the LB broth.



I'm assuming now that my cell stock mightnt be too viable and might start again.
(I have very little construct left.
My protocol for transformation was to take 50ug of the construct and add it to 50ul of electrocompetent ecoli cells.
I mixed gently and incubated on ice for 30 minutes. I heat shocked the cells for 30 seconds at 42C and then put them on ice for 2 mins.
That was when i added 500ul of SOC.I put the tubes on a shaker for 30 minutes at 37C, then plated them.)


Does anyone have a protocol for transforming a plasmid into ecoli cells, or would it be the same as how i did it for the construct?


I might try a last ditch attempt to throw all the glycerol stocks into LB broth and keep them shaking at 37C for 48 hours to see if i can get anything again.

Thanks for the help so far everyone.

#6 molstudent

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Posted 04 July 2010 - 09:18 PM

50ul cell and 50ul og glycerol seems very little but the ratio is fine. i usually do 500ul of each. for this ratio you should have scrapped the cells off instead of thawing it, thawing it may have killed your cells.

your transformation protocol seems fine but i incubate in SOC for 1 hour. heres how mine usually goes:

-add some plasmid to competent cells
-leave on ice for 5 minutes
-heat shock for 90secs
-ice for 2 min
-add 250ul SOC and shake at 37C for 1 hour

#7 cm13

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Posted 05 July 2010 - 03:30 AM

50ul cell and 50ul og glycerol seems very little but the ratio is fine. i usually do 500ul of each. for this ratio you should have scrapped the cells off instead of thawing it, thawing it may have killed your cells.

your transformation protocol seems fine but i incubate in SOC for 1 hour. heres how mine usually goes:

-add some plasmid to competent cells
-leave on ice for 5 minutes
-heat shock for 90secs
-ice for 2 min
-add 250ul SOC and shake at 37C for 1 hour


How much plasmid should i use? should i go with about 50ug?
Also, would 250ul SOC be enough, considering before i would use 500ul?

#8 virusfan

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Posted 05 July 2010 - 06:18 AM

[/quote]

1) I went with 50ul of the cells and 50ul of Glycerol. Would this have been too much, or not enough?
2) I'm pretty sure that the plasmid i ended up getting was the correct one.

3) I didnt let the stock thaw out fully, but at the same time i did let it thaw a little instead of scraping off the cells.

4)Sorry, i forgot to add that i let the transformed cells grow in SOC broth. This is what i plated out first. I grew colonies from the plates, and took a colony and grew it up in the LB broth.



I'm assuming now that my cell stock mightnt be too viable and might start again.
(I have very little construct left.
My protocol for transformation was to take 50ug of the construct and add it to 50ul of electrocompetent ecoli cells.
I mixed gently and incubated on ice for 30 minutes. I heat shocked the cells for 30 seconds at 42C and then put them on ice for 2 mins.
That was when i added 500ul of SOC.I put the tubes on a shaker for 30 minutes at 37C, then plated them.)


Does anyone have a protocol for transforming a plasmid into ecoli cells, or would it be the same as how i did it for the construct?


I might try a last ditch attempt to throw all the glycerol stocks into LB broth and keep them shaking at 37C for 48 hours to see if i can get anything again.

Thanks for the help so far everyone.
[/quote]

Hi there,
Your transformation method looks fine, but I am confused. Why??? The reason is you mentioned that you are using electrocompetent E. coli cells, but you were not doing electroporation. I am doubt if that is possible. Personally, I have not tried that before.

As mentioned previously by phage434, I totally agreed that probably you did not vortex your stock to mix your bacteria with the glycerol well. Hence, you missed the area with bacteria.

If your bacterial stocks are very precious, always leave your stocks on dry ice, never allow them to defroze completely. Just scrap some of the stock when you need to streak them onto agar plate.

Hope this helps. :D

Edited by virusfan, 05 July 2010 - 06:18 AM.


#9 Lapsang

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Posted 06 July 2010 - 10:02 AM

My protocol for transformation was to take 50ug of the construct and add it to 50ul of electrocompetent ecoli cells.
I mixed gently and incubated on ice for 30 minutes. I heat shocked the cells for 30 seconds at 42C and then put them on ice for 2 mins.
That was when i added 500ul of SOC.I put the tubes on a shaker for 30 minutes at 37C, then plated them.)


Does anyone have a protocol for transforming a plasmid into ecoli cells, or would it be the same as how i did it for the construct?


My transformation protocol in DH5alpha E. coli only uses 10ng of plasmid (not sure if you mean 50ng rather than 50ug though) for 50ul of cells. Other than that, our protocols are about the same, though I heat shock at 37, not 42 for DH5alpha and shake for 1 hour, not 30 mins (but I don't think that would matter much...)

#10 cm13

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Posted 07 July 2010 - 02:46 AM

My protocol for transformation was to take 50ug of the construct and add it to 50ul of electrocompetent ecoli cells.
I mixed gently and incubated on ice for 30 minutes. I heat shocked the cells for 30 seconds at 42C and then put them on ice for 2 mins.
That was when i added 500ul of SOC.I put the tubes on a shaker for 30 minutes at 37C, then plated them.)


Does anyone have a protocol for transforming a plasmid into ecoli cells, or would it be the same as how i did it for the construct?


My transformation protocol in DH5alpha E. coli only uses 10ng of plasmid (not sure if you mean 50ng rather than 50ug though) for 50ul of cells. Other than that, our protocols are about the same, though I heat shock at 37, not 42 for DH5alpha and shake for 1 hour, not 30 mins (but I don't think that would matter much...)



sorry, typo. I meant 50ng.
I'll try that so. Thanks.

#11 cm13

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Posted 12 July 2010 - 02:29 AM

I've a follow-up question.

I found at the back of the fridge that i had kept the cells i made spread plates with before. If i have these ecoli bacterial cells on an agar plate, how long will they last in a fridge?
There's kanamycin antibiotic added to the plate, but the plate wasnt covered in tinfoil, so that might be degraded by now.
The plate still looks ok though. im just wondering if the cells would be viable or useful by now.

#12 fysio lab

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Posted 13 July 2010 - 01:44 AM

I've a follow-up question.

I found at the back of the fridge that i had kept the cells i made spread plates with before. If i have these ecoli bacterial cells on an agar plate, how long will they last in a fridge?
There's kanamycin antibiotic added to the plate, but the plate wasnt covered in tinfoil, so that might be degraded by now.
The plate still looks ok though. im just wondering if the cells would be viable or useful by now.


Try to grow up some colonies in LB with fresh kanamycin added...if you see growth they survived. Hopefully your plate isn't dried up totally (agar being like a chip) otherwise your bacteria will certainly be dead.
Good luck

#13 cm13

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Posted 14 July 2010 - 02:14 AM

So to follow up on this:

1) I tried transforming my bacteria with my plasmid sample. My protocol was as i mentioned earlier. With 50ng of plasmid and 50ul of cells. I also changed the amount of SOC added to 1ml instead of 500ul. Having plated this, i left the plates at 37C overnight. There is nothing on the plates. Should i maybe leave it for another day?

2)I also found i had plates from the last time i made the plasmid. However these plates are 4 months old and were kept in the fridge. I attempted to take a colony from the plate, add it to 5mls of LB broth, and add 5ul of Kanamycin. (Again at a concentration of 30mg/ml).
Leaving this starter culture for 8 hours rotating at 37C , i found nothing. I left it overnight at the same conditions and there was still nothing.

Should i maybe try step 2 again, but without the kanamycin? This might allow for growth of the cells.

#14 Mycobacterial Madman

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Posted 19 July 2010 - 10:38 PM

like the others said, try both of these:

heat shock transformation (my protocol)

thaw chemically competent cells on ice
add 1uL of plasmid (around 10 ng is fine) and mix gently with pipette
leave cells on ice for 30 min
heat shock cells @ 42 degrees for 2 min
leave cells on ice for 5 min
add 900uL SOC / LB, recover @ 37oC with shaking for 1 hour.
spin down in microfuge for 2 min at max speed, resuspend pellet in 100uL
plate 100uL resuspension on agar with appropriate antibiotic, incubate 37 degrees overnight.

make sure to include a control transformation of another plasmid, so you know your compentent cells are working.

last shot recovery of glycerol stock:

innoculate entire glycerol stock in 3mL LB/SOC @ 37 degrees for a couple hours
as before spin down to collect pellet and resuspend in 100uL
plate on agar with appropriate antibiotic, also re-use the tube with LB/SOC + antibiotic and incubate @ 37 overnight.

remember to make multiple glycerol freezer stocks next time, I use 400uL of 50% glycerol and 600uL of culture.

the plate in the fridge could still be viable as well if its not dried out completely, so try innoculating big loopfuls into both media and media + antibiotic.

good luck!

#15 cm13

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Posted 20 July 2010 - 01:12 AM

Thank you everyone for the help.
I managed to get my plasmids growing properly in the end after transforming in my old plasmid into the ecoli cells.
I also made up new Agar plates with the colonies, and new glycerol stocks, so i should be ok for regrowing in the future.

Thanks once again!




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