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please help!!cloning problems..


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#1 yang

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Posted 16 June 2010 - 09:33 AM

hi,
i am fresh to molecular biology and recently joined a lab. My PI asked me to do some cloning stuff under the guidance of post doc. I did every thing according to his suggestions but now facing a problem and my post doc is on vacation.
I was asked to clone some plasmid with GFP gene and I did with the help of restriction digestion. I used ligase to ligate the genes and plated them on a LB plate with tetR since the plasmids have tet resistance. I found around 16 clonies whicg are green and grew on tet. I took these colonies and grew them on LB medium and they grew well and they look green. I also checked for fluorescence and my post doc said they are fluorescing. when I try to grow them in tet+lb, their growth is very slow. When I try to subculture, the cells are not growing in LB+ tet. only in lb, they are not green. I do not what happened but I think they lost the plasmid. Is there any way I can get the plasmid back or make them grow well. if my pi knows abt this, he will kick me out of the lab. plz tell me wht i need to do...i need this asap...

i am very tensed..pls help....

#2 lsek

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Posted 17 June 2010 - 05:50 AM

Hi,

I'm assuming that you are working on bacteria (E. coli).

Did you keep a glycerol stock of each of the transformant in -80C? If yes, just streak a fresh one and grow again in LB Tet broth.
If you don't have glycerol stock, re-trace the LB tet agar plate and streak a little of a colony (you had 16 right?) and grow in LB tet broth.

Colony (bacteria) on plate have a shelf life of roughly two month (or less), whereas bacteria in a broth have lesser. In order to preserve the viability of bacteria, you will always have to re-plate or re-culture. The best way to preserve is still glycerol stock.

Good luck.

===><===


#3 yang

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Posted 17 June 2010 - 10:21 AM

Hi,

I'm assuming that you are working on bacteria (E. coli).

Did you keep a glycerol stock of each of the transformant in -80C? If yes, just streak a fresh one and grow again in LB Tet broth.
If you don't have glycerol stock, re-trace the LB tet agar plate and streak a little of a colony (you had 16 right?) and grow in LB tet broth.

Colony (bacteria) on plate have a shelf life of roughly two month (or less), whereas bacteria in a broth have lesser. In order to preserve the viability of bacteria, you will always have to re-plate or re-culture. The best way to preserve is still glycerol stock.

Good luck.


Thanks for the information and the bacteria I am working on is something called dh5 alpha. I prepared glycerol stocks but now all the cells are dead. I tried growing them on LB medium but none grew. The plate from which I had the original colonies is also not helping because they do not grow again even on LB medium or on plates. Do you think I should do the whole process again.
Thanks in advance.

Edited by yang, 17 June 2010 - 10:23 AM.


#4 Michaelro

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Posted 19 June 2010 - 07:06 AM

Hi
I think that your problem was growing the bacteria in Lb without antibiotics after obtaining the colonies.
Unless the selection pressure is applied, bacteria will lose the plasmid.
Maybe you should try to repeat the ligation and after having the colonies, grow them in Lb wih antibiotics .
Furthermore, You will probably do a miniprep to check that your plasmid is OK.
Once You have the plasmid DNA, even if You lose the bacteria, You wll always be able to transform fresh bacterial stock.
Hope it is helpful
Good Luck
Michael

Hi,

I'm assuming that you are working on bacteria (E. coli).

Did you keep a glycerol stock of each of the transformant in -80C? If yes, just streak a fresh one and grow again in LB Tet broth.
If you don't have glycerol stock, re-trace the LB tet agar plate and streak a little of a colony (you had 16 right?) and grow in LB tet broth.

Colony (bacteria) on plate have a shelf life of roughly two month (or less), whereas bacteria in a broth have lesser. In order to preserve the viability of bacteria, you will always have to re-plate or re-culture. The best way to preserve is still glycerol stock.

Good luck.


Thanks for the information and the bacteria I am working on is something called dh5 alpha. I prepared glycerol stocks but now all the cells are dead. I tried growing them on LB medium but none grew. The plate from which I had the original colonies is also not helping because they do not grow again even on LB medium or on plates. Do you think I should do the whole process again.
Thanks in advance.



#5 yang

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Posted 21 June 2010 - 06:03 AM

Oh, I did not know this. So this means that I should start the process again. Okay. Thanks for the information, You guys were lot of help.




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