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Help with cloning!.


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#16 dnarna909

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Posted 11 May 2009 - 05:04 PM

I had the same problem.
Then I change into the low melt agrose and use new competent cell , my vector is pcDNA3.1, it works.
Now I am trying to ligation a 2kb fragment into pAdTrack-CMV vector which is kanamycin resistence, I can only get a few clone, and these clone turn out to be no plasmid containing.

I keep the same agrose I used before,so as the competent cell and ligation buffer, I don't know why the clone I got can not be lysis by lysis buffer, there is no problem with the buffer.

#17 HomeBrew

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Posted 11 May 2009 - 06:40 PM

I think we're focussing on the wrong thing here -- everyone in my lab routinely gel purifies digested fragments and recovers them with the Qiagen kit, and has for years, and cloning works fine. I just did a transformation yesterday from DNA thus prepared and got 120 colonies. I don't think your problem is caused by the Qiagen kit...

Thanks for the posting and to answer your questions, I cut my (insert the 2Kb fragment with Sma I and also tried Xma I as well just cos of the blunt end and the sticky end issues) and I cut the back bone again with Sma I . There is a Pac I site between the two Sma I sties in the backbone as well. and I used Sma I and Pac I just to ensure that I get the proper fragments and always purify the highest band the backbone part that Im interested in. The plasmid that I cut the 2Kb fragment out of is similar to the backbone plamid except for different promoter. But I need to somehow move it to the interested backbone inorder for the experiments.


If I understand what you're saying above correctly (it's a bit confusing trying to keep track of backbones), you're trying to clone a piece of one vector into another vector. This can be very problematic, as you may be introducing machinery from one vector into the other such that the ultimate plasmid clone can no longer replicate or partition correctly due to incompatible genes competing with one another (those that existed on the vector to begin with coupled with those that are coming in on the 2 Kb piece).

Coupled with blunt-end digestion, this is going to be difficult. Do you know exactly what's on the 2 Kb piece you're trying to clone?

#18 micky74

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Posted 12 May 2009 - 08:02 AM

I think we're focussing on the wrong thing here -- everyone in my lab routinely gel purifies digested fragments and recovers them with the Qiagen kit, and has for years, and cloning works fine. I just did a transformation yesterday from DNA thus prepared and got 120 colonies. I don't think your problem is caused by the Qiagen kit...

Thanks for the posting and to answer your questions, I cut my (insert the 2Kb fragment with Sma I and also tried Xma I as well just cos of the blunt end and the sticky end issues) and I cut the back bone again with Sma I . There is a Pac I site between the two Sma I sties in the backbone as well. and I used Sma I and Pac I just to ensure that I get the proper fragments and always purify the highest band the backbone part that Im interested in. The plasmid that I cut the 2Kb fragment out of is similar to the backbone plamid except for different promoter. But I need to somehow move it to the interested backbone inorder for the experiments.


If I understand what you're saying above correctly (it's a bit confusing trying to keep track of backbones), you're trying to clone a piece of one vector into another vector. This can be very problematic, as you may be introducing machinery from one vector into the other such that the ultimate plasmid clone can no longer replicate or partition correctly due to incompatible genes competing with one another (those that existed on the vector to begin with coupled with those that are coming in on the 2 Kb piece).

Coupled with blunt-end digestion, this is going to be difficult. Do you know exactly what's on the 2 Kb piece you're trying to clone?


Do they follow any trick to get the band out of the gel? Do they check the abs of the recovered pcr product by UV nanodrop or other? Do they smell a bit of ethanol after? Do they run another ethanol precipitation after that or from agarose extraction they go straight to ligation? What is the volume of the ligation reaction and the maximum amount of PCR extracted (5- 10 -20 -50% of total volume?)

Cheers

#19 HomeBrew

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Posted 12 May 2009 - 10:46 AM

Do they follow any trick to get the band out of the gel?


No.

Do they check the abs of the recovered pcr product by UV nanodrop or other?


No.

Do they smell a bit of ethanol after?


No, but we dry the Qiagen columns with a two minute spin before elution.

Do they run another ethanol precipitation after that or from agarose extraction they go straight to ligation?


Straight to ligation.

What is the volume of the ligation reaction and the maximum amount of PCR extracted (5- 10 -20 -50% of total volume?)


We do our ligations in 20 ul, using the Ready-To-Go T4 DNA Ligase from GE Healthcare. I don't know what the percent recovery is; we never bother to check it.

We also use chemically competent cells we make ourselves using a RbCl2 method.

We usually get our clones with a minimum of fuss -- failure to get a correct clone is not unheard of in the lab, but it is somewhat rare...

#20 micky74

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Posted 12 May 2009 - 11:01 AM

Do they follow any trick to get the band out of the gel?


No.

Do they check the abs of the recovered pcr product by UV nanodrop or other?


No.

Do they smell a bit of ethanol after?


No, but we dry the Qiagen columns with a two minute spin before elution.

Do they run another ethanol precipitation after that or from agarose extraction they go straight to ligation?


Straight to ligation.

What is the volume of the ligation reaction and the maximum amount of PCR extracted (5- 10 -20 -50% of total volume?)


We do our ligations in 20 ul, using the Ready-To-Go T4 DNA Ligase from GE Healthcare. I don't know what the percent recovery is; we never bother to check it.

We also use chemically competent cells we make ourselves using a RbCl2 method.

We usually get our clones with a minimum of fuss -- failure to get a correct clone is not unheard of in the lab, but it is somewhat rare...


So you do not check for the vector/insert ratio? Or you check by visual inspection on a gel? How many ng of vectors more or less for each ligation?
At the end you run also vector alone and vector= insert and you alway obtain more colonies on the vector=insert or you do not bother to check that?

Cheers

#21 HomeBrew

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Posted 12 May 2009 - 05:43 PM

So you do not check for the vector/insert ratio? Or you check by visual inspection on a gel? How many ng of vectors more or less for each ligation?
At the end you run also vector alone and vector= insert and you alway obtain more colonies on the vector=insert or you do not bother to check that?


I very rarely check for ratios, but I usually have a rough idea how much insert I have by the intensity of the band produced by the PCR. Ditto for the vector. We no longer bother to do vector alone controls. We don't even own a spec, though there's a nanodrop in a neighboring lab, should the need arise.

As I've stated before in other threads (and I know it's heresy around here), such things (in our hands, anyway) are not critical -- cloning in the lab has become routine -- we usually get what we're looking for on the first or second try. I'm not saying there's never a difficult clone, just that it's the exception, not the rule.

The only things we do that are perhaps different than other labs is to use guanosine in TAE to cast and run gels from which we're going to recover fragments (to protect the DNA from UV damage), and use the glassified Ready-to-Go ligase linked to above.




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