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Help with cloning!.


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#1 molecule

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Posted 10 May 2009 - 01:03 PM

For the past three months I have been trying to clone a 2KB fragment ( cut out from a 7kb plasmid) to a 12kb back bone. I have failed each time with the cloning, due to a number of reasons, since how ever much I try it seems that I can’t get a single colony after the transformation. I have good recovery of DNA after the purifications, competent cells are good.
Lately It seems that the gel purification step is some what inhibiting the ligations which became evident after I did some controls with a known plasmid ( just by linearising the plasmid and by trying to religate them)
I was wondering if any one could advice me on this? Could the gel purification some how inhibit the ligation/ transformations? Has anyone experienced this before? Are there any other alternative methods to isolate the fragment that I’m interested in other than using a gel purification/ if not other ways to clean the fragment cut off the gel ( I have tried the kits as well as the traditional methods of ethanol prepping and there is no difference) Any advice or information would be greatly appreciated!.

Thanks,

#2 HomeBrew

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Posted 10 May 2009 - 02:54 PM

What enzyme(s) are you using? What is the nature of the 2 Kb fragment you're cutting out of another plasmid? Is it part of the plasmid, or an insert carried by a plasmid?

When you linearizing the plasmid and religated as a control, did you gel purify this fragment? If not, how do you know that gel purification is the issue? You could have just recovered transformants derived from uncut plasmid in your digest...

The fact that you're getting no colonies at all is troubling... Are you sure your ligation reaction is working? How did you prepare your vector DNA?

When you are cutting bands out of your gel, do you try to minimize exposure to UV light? When we do this in my lab, we add guanosine to the gel and the running buffer if we're going to cut a band out; it acts as a UV protectant (see Gründemann, D., and E. Schömig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. Biotechniques 21(5):898-903.).

#3 molecule

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Posted 10 May 2009 - 08:58 PM

What enzyme(s) are you using? What is the nature of the 2 Kb fragment you're cutting out of another plasmid? Is it part of the plasmid, or an insert carried by a plasmid?

When you linearizing the plasmid and religated as a control, did you gel purify this fragment? If not, how do you know that gel purification is the issue? You could have just recovered transformants derived from uncut plasmid in your digest...

The fact that you're getting no colonies at all is troubling... Are you sure your ligation reaction is working? How did you prepare your vector DNA?

When you are cutting bands out of your gel, do you try to minimize exposure to UV light? When we do this in my lab, we add guanosine to the gel and the running buffer if we're going to cut a band out; it acts as a UV protectant (see Gründemann, D., and E. Schömig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. Biotechniques 21(5):898-903.).



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 I’m 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.

When leanarizing yes, I did gel purify the fragment. And earlier I dried doing it W/O gel purifying, just ethanol prepping the digest since it is the leaner fragment I want. and when I did the ethanol prepe, ligated and transformed i got a colonies and when i did the gel purification and do the same experiment I dont get colonies. whihc was the thing that made me think that gel purification was inhibiting. For the control I used a plasmid that could be used in a Blue white selection to make things more evitable.

I don’t understand what you mean by how did you prepare the vector, but in the case of my ( experimental not the control that I talked about before) I always phospahatase treat the vector portion to ensure that there is no self ligation.
Yes I try to mininize the exposure to UV and cut the bands as soon as I can, but I have not paid a lot of attention to clean sides of the cut piece to minimize the agarose. Could that be a big issue. And I used a 1% gel.

Edited by molecule, 10 May 2009 - 09:01 PM.


#4 Vini

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Posted 10 May 2009 - 09:44 PM

What enzyme(s) are you using? What is the nature of the 2 Kb fragment you're cutting out of another plasmid? Is it part of the plasmid, or an insert carried by a plasmid?

When you linearizing the plasmid and religated as a control, did you gel purify this fragment? If not, how do you know that gel purification is the issue? You could have just recovered transformants derived from uncut plasmid in your digest...

The fact that you're getting no colonies at all is troubling... Are you sure your ligation reaction is working? How did you prepare your vector DNA?

When you are cutting bands out of your gel, do you try to minimize exposure to UV light? When we do this in my lab, we add guanosine to the gel and the running buffer if we're going to cut a band out; it acts as a UV protectant (see Gründemann, D., and E. Schömig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. Biotechniques 21(5):898-903.).



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 I’m 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.

When leanarizing yes, I did gel purify the fragment. And earlier I dried doing it W/O gel purifying, just ethanol prepping the digest since it is the leaner fragment I want. and when I did the ethanol prepe, ligated and transformed i got a colonies and when i did the gel purification and do the same experiment I dont get colonies. whihc was the thing that made me think that gel purification was inhibiting. For the control I used a plasmid that could be used in a Blue white selection to make things more evitable.

I don’t understand what you mean by how did you prepare the vector, but in the case of my ( experimental not the control that I talked about before) I always phospahatase treat the vector portion to ensure that there is no self ligation.
Yes I try to mininize the exposure to UV and cut the bands as soon as I can, but I have not paid a lot of attention to clean sides of the cut piece to minimize the agarose. Could that be a big issue. And I used a 1% gel.



I dont believe that gel purification step would interfere with ligations. We religiously do this in our lab and we dont have problems because of this. DNA used for ligations should be free of any other contaminants like protein, salts and ethanol that might interfere with ligase reaction. What is funny is the fact that you are not even getting religations (that would be the case if phosphatase treatment is not working). Are u sure that your ligase buffer and enzyme are working fine? I have doubts regarding this in my mind.

#5 molecule

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Posted 10 May 2009 - 09:54 PM

I have tested the enzymes and the buffers, and also have started a new batch of ligation buffers and T4 ligase from NEB since it was a suspect that they were contaminated!. So I believe they seem to be working well. Do you have any ideas as to how I could re-do my experiments, or any other methods to test to confirm these?

Edited by molecule, 10 May 2009 - 09:55 PM.


#6 Vini

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Posted 10 May 2009 - 10:04 PM

I have tested the enzymes and the buffers, and also have started a new batch of ligation buffers and T4 ligase from NEB since it was a suspect that they were contaminated!. So I believe they seem to be working well. Do you have any ideas as to how I could re-do my experiments, or any other methods to test to confirm these?


hey molecule,

are u saying that u r not getting colonies even after using the fresh batch of enzyme n buffer???? :unsure:

#7 micky74

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Posted 10 May 2009 - 10:35 PM

For the past three months I have been trying to clone a 2KB fragment ( cut out from a 7kb plasmid) to a 12kb back bone. I have failed each time with the cloning, due to a number of reasons, since how ever much I try it seems that I can’t get a single colony after the transformation. I have good recovery of DNA after the purifications, competent cells are good.
Lately It seems that the gel purification step is some what inhibiting the ligations which became evident after I did some controls with a known plasmid ( just by linearising the plasmid and by trying to religate them)
I was wondering if any one could advice me on this? Could the gel purification some how inhibit the ligation/ transformations? Has anyone experienced this before? Are there any other alternative methods to isolate the fragment that I’m interested in other than using a gel purification/ if not other ways to clean the fragment cut off the gel ( I have tried the kits as well as the traditional methods of ethanol prepping and there is no difference) Any advice or information would be greatly appreciated!.

Thanks,



I do have a similar problem. So I skipped right now the gel purification. Which kit are you using for gel extraction?I read somewhere in another forum that Qiagen has added some chemical in the QG solution that seems to disturb ligation......if anyone has a feedback on that would be appreciated
Well if you think the gel extraction can be the problem try to skip it

Cheers

#8 molecule

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Posted 10 May 2009 - 11:27 PM

Hi DRN and micky,

Yes I have had problems even after using the new batch of buffers etc. I have being using the Quigen kits for the gel purifications. Well but it seems that I cant skip the gel purification step, since I cant think of any other way to get my desired fragments after the restriction enzyme digest with Sma I on my plasmids. Or is there any other way to get around with it which I might not know???

Edited by molecule, 10 May 2009 - 11:29 PM.


#9 micky74

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Posted 10 May 2009 - 11:43 PM

Hi DRN and micky,

Yes I have had problems even after using the new batch of buffers etc. I have being using the Quigen kits for the gel purifications. Well but it seems that I cant skip the gel purification step, since I cant think of any other way to get my desired fragments after the restriction enzyme digest with Sma I on my plasmids. Or is there any other way to get around with it which I might not know???



Ok just got to the lab

Here is my update. So using gel extraction I got few colonies on control, no colonies at all in insert+vector.

Using only the quiagen kit for PCR purification :no colonies on the vector treated with SAP alone. Around 60-80 clonies on 3:1 and 5:1 pcr:vector I prepared. So actually it made a huge difference. Now the vector was treated with SAP and digested with 2 different enzymes so I do expect most of my vector to contain an insert.

Guess the difference could be due to: or no exposure of DNA to UV or Qiagen gel extraction kit solutions. I can not rule out which one of course.

About your problem. I read that the problems come with the qiagen kit as they changed the Qg buffer somewhere. WHat about try another kit from another company? It could be even the agarose inhibithing your reaction, but I do think could be the 1st buffer. I remember this post I read the writer was actually a PI that observed a huge difference in cloning efficiency before and after a new patch of Qiagen kit. He wrote he phoned to Qiagen that admitted to have changed the composition of buffer QG to make it more active against agarose. The PI said he went back to low melt agarose and ligation started to work again.

So if you can't skip the gel extraction try another kit, try low melting and minimize exposure to UV for a matter of seconds, that is the best solution I can give to you


Cheers

Edited by micky74, 10 May 2009 - 11:52 PM.


#10 gsatan

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Posted 10 May 2009 - 11:52 PM

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

#11 molecule

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Posted 10 May 2009 - 11:56 PM

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??

#12 micky74

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Posted 10 May 2009 - 11:56 PM

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.

#13 micky74

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Posted 11 May 2009 - 12:01 AM

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

#14 molecule

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Posted 11 May 2009 - 12:27 AM

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

#15 micky74

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Posted 11 May 2009 - 12:38 AM

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




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