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Some colonies, but not enough...


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#1 303microbialist

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Posted 18 June 2009 - 09:27 AM

So, I've been using the old Topo TA Original kit by Invitrogen to clone 16/18s PCR amplicons (1.40-1.8 Kb). Pretty straight forward, but for some reason I cannot get this to consistently work at a reasonable efficiency. When it works, I get a couple hundred white colonies per plate, plus a handful of blues--but a lot of times I'll only get like 20-30 colonies per plate (or less). Never no colonies though. If I go back and re-transform with my left over ligation reaction (heat shock Fprime cells at 42 for 30 sec), sometimes I get the plethora of colonies I need, and sometimes not. If not, I just go back and re-ligate with left over DNA... If that dosen't work, then I start all over. This makes it seem like my issue is with the transformation, but of course I can't be sure. So each little project has turned into a frustrating series of what seem like random trials until I get enough colonies to harvest, and I'm hoping I can draw on some of your wisdom to make this stuff work better.

Thanks.

#2 eldon

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Posted 18 June 2009 - 10:08 AM

So, I've been using the old Topo TA Original kit by Invitrogen to clone 16/18s PCR amplicons (1.40-1.8 Kb). Pretty straight forward, but for some reason I cannot get this to consistently work at a reasonable efficiency. When it works, I get a couple hundred white colonies per plate, plus a handful of blues--but a lot of times I'll only get like 20-30 colonies per plate (or less). Never no colonies though. If I go back and re-transform with my left over ligation reaction (heat shock Fprime cells at 42 for 30 sec), sometimes I get the plethora of colonies I need, and sometimes not. If not, I just go back and re-ligate with left over DNA... If that dosen't work, then I start all over. This makes it seem like my issue is with the transformation, but of course I can't be sure. So each little project has turned into a frustrating series of what seem like random trials until I get enough colonies to harvest, and I'm hoping I can draw on some of your wisdom to make this stuff work better.

Thanks.


short answer: you only need one colony.


are your cells one shot or homemade one shot? or are they competent cells that you can re-use (e.g., 200uL of cells/tube)...these cells can give inconsistent results from freeze/thaw

#3 303microbialist

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Posted 18 June 2009 - 01:11 PM

i just use one shot e coli, non-homemade.

the whole problem is i actually need 95-190 colonies per reaction. i'm doing environmental sampling so each colony represents a population sample, identifying only 1 of 10s of thousands of phyotypes that exist in my template DNA.


So, I've been using the old Topo TA Original kit by Invitrogen to clone 16/18s PCR amplicons (1.40-1.8 Kb). Pretty straight forward, but for some reason I cannot get this to consistently work at a reasonable efficiency. When it works, I get a couple hundred white colonies per plate, plus a handful of blues--but a lot of times I'll only get like 20-30 colonies per plate (or less). Never no colonies though. If I go back and re-transform with my left over ligation reaction (heat shock Fprime cells at 42 for 30 sec), sometimes I get the plethora of colonies I need, and sometimes not. If not, I just go back and re-ligate with left over DNA... If that dosen't work, then I start all over. This makes it seem like my issue is with the transformation, but of course I can't be sure. So each little project has turned into a frustrating series of what seem like random trials until I get enough colonies to harvest, and I'm hoping I can draw on some of your wisdom to make this stuff work better.

Thanks.


short answer: you only need one colony.


are your cells one shot or homemade one shot? or are they competent cells that you can re-use (e.g., 200uL of cells/tube)...these cells can give inconsistent results from freeze/thaw



#4 Zhongbo

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Posted 18 June 2009 - 06:39 PM

I never use this kit and have no knowledge about the procedure but my suggestion is
1, make more plates for each transformation. For example, 5 or more plates for each ligation-transforamtion.
2, try to decrease antibiotic concentration while set a control to make sure cell does not grow without transformation.
3, check your transformation procedure. Is the heat-shock temperature real? what kind of tubes are you using to perform heat-shock? How long is the recovery time?

So, I've been using the old Topo TA Original kit by Invitrogen to clone 16/18s PCR amplicons (1.40-1.8 Kb). Pretty straight forward, but for some reason I cannot get this to consistently work at a reasonable efficiency. When it works, I get a couple hundred white colonies per plate, plus a handful of blues--but a lot of times I'll only get like 20-30 colonies per plate (or less). Never no colonies though. If I go back and re-transform with my left over ligation reaction (heat shock Fprime cells at 42 for 30 sec), sometimes I get the plethora of colonies I need, and sometimes not. If not, I just go back and re-ligate with left over DNA... If that dosen't work, then I start all over. This makes it seem like my issue is with the transformation, but of course I can't be sure. So each little project has turned into a frustrating series of what seem like random trials until I get enough colonies to harvest, and I'm hoping I can draw on some of your wisdom to make this stuff work better.

Thanks.



#5 jiajia1987

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Posted 18 June 2009 - 07:36 PM

So, I've been using the old Topo TA Original kit by Invitrogen to clone 16/18s PCR amplicons (1.40-1.8 Kb). Pretty straight forward, but for some reason I cannot get this to consistently work at a reasonable efficiency. When it works, I get a couple hundred white colonies per plate, plus a handful of blues--but a lot of times I'll only get like 20-30 colonies per plate (or less). Never no colonies though. If I go back and re-transform with my left over ligation reaction (heat shock Fprime cells at 42 for 30 sec), sometimes I get the plethora of colonies I need, and sometimes not. If not, I just go back and re-ligate with left over DNA... If that dosen't work, then I start all over. This makes it seem like my issue is with the transformation, but of course I can't be sure. So each little project has turned into a frustrating series of what seem like random trials until I get enough colonies to harvest, and I'm hoping I can draw on some of your wisdom to make this stuff work better.

Thanks.


How do you add the ligated reactions to the cells? Just eject the ligated reactions into the solution of cells and swirl it once. Pipetting will kill the cells or make it fragile during transformation. This usually gives me more than 100 colonies and sometimes up to 200 colonies.

#6 microgirl

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Posted 19 June 2009 - 05:13 AM

Here's the expensive answer - there's an Invitrogen kit (can't remember what the part # is) but it's the TOPO TA cloning kit for sequencing with TOP10 chemically competent cells and the PCR4TOPO vector. It sounds like you want to use it for the same thing I did - you get hundreds of colonies per rxn if you plate the whole thing. You can try splitting your cells (keep them on ice and refreeze your cell prep) and only use half the vector amount to get more rxns for the price.

#7 303microbialist

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Posted 19 June 2009 - 08:39 AM

thanks everyone! I transformed again last night, using left over ligation reaction, spread 60 ul on 4 plates and woke up this morning to at least 300 hundred white colonies. always a good way to start the day...

I did everything exactly the same (I'm always gentle with cells when adding the plasmid), so it's still a bit of mystery...

#8 eldon

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Posted 20 June 2009 - 07:51 AM

thanks everyone! I transformed again last night, using left over ligation reaction, spread 60 ul on 4 plates and woke up this morning to at least 300 hundred white colonies. always a good way to start the day...

I did everything exactly the same (I'm always gentle with cells when adding the plasmid), so it's still a bit of mystery...


so it comes down to your spreader being too hot...unless you use glass beads.




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