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Help with cloning!. - Cloning problems (May/10/2009 )

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Hi Micky, Thanks for the responce! So in my case after I digest with Smal I my vector and Insert is there any other way other than gel purification to purify the sample? ( Sorry if I;m just confused! ), cos I need a 2kB insert which is a result of the Sma I digest to be inserted to a back bone of about 10kB which is also digested with Sma I. I'm just confused on the step of purifying my desired band?
- and if I understood you correct do you mean that I should try just PCR my desired insert? and then try to go on with the ligation??

-molecule-

gsatan on May 10 2009, 11:52 PM said:

I believe that gel purification step wouldn't interfere with ligations.



Well as I said. Starting with same material. and differing only in one step: purification at the end or using qiagen PCR purification or running a gel using then qiagen extraction kit from agarose. I got a huge difference and the colonies are growing only in vector and insert purified with Qiagen PCR purification. In this experiment I have no colonies on the vector alone.

-micky74-

molecule on May 10 2009, 11:56 PM said:

Hi Micky, Thanks for the responce! So in my case after I digest with Smal I my vector and Insert is there any other way other than gel purification to purify the sample? ( Sorry if I;m just confused! ), cos I need a 2kB insert which is a result of the Sma I digest to be inserted to a back bone of about 10kB which is also digested with Sma I. I'm just confused on the step of purifying my desired band?
- and if I understood you correct do you mean that I should try just PCR my desired insert? and then try to go on with the ligation??



If I can understand well. You have a 2KB insert in one vector you want to insert in another vector digested with SmaI?

Or you cut from the first vector, gel purify using low melt agarose and go on with ligation, or yes you can PCR your insert with a pfx (a polymerase prro reading that is going to give you a bulnt PCR product), purify the PCR product (or not, ask for advice here) and go on with ligation. For the other vector, cut with SmaI, treat with SAP or CIAP, heat inactivate, purify and use for ligation

-micky74-

Thanks Micky. I will try this and see if this will work!.

-molecule-

molecule on May 11 2009, 12:27 AM said:

Thanks Micky. I will try this and see if this will work!.



I'm not sure I avoid blunt end, but I guess you should phosphorilate your PCR product, please look at the topic around for blunt cloning

Micky

-micky74-

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.

-dnarna909-

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...

molecule on May 11 2009, 12:58 AM said:

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?

-HomeBrew-

HomeBrew on May 11 2009, 06:40 PM said:

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...

molecule on May 11 2009, 12:58 AM said:

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

-micky74-

micky74 on May 12 2009, 12:02 PM said:

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


No.

micky74 on May 12 2009, 12:02 PM said:

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


No.

micky74 on May 12 2009, 12:02 PM said:

Do they smell a bit of ethanol after?


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

micky74 on May 12 2009, 12:02 PM said:

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


Straight to ligation.

micky74 on May 12 2009, 12:02 PM said:

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...

-HomeBrew-

HomeBrew on May 12 2009, 10:46 AM said:

micky74 on May 12 2009, 12:02 PM said:

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


No.

micky74 on May 12 2009, 12:02 PM said:

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


No.

micky74 on May 12 2009, 12:02 PM said:

Do they smell a bit of ethanol after?


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

micky74 on May 12 2009, 12:02 PM said:

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


Straight to ligation.

micky74 on May 12 2009, 12:02 PM said:

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

-micky74-
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