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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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ligation problems
Posted by: nvergara (IP Hidden, Unregistered user, )
Date: April 18, 2005 04:45AM
Dear all,
I'm completely usuccessful in ligating a NcoI/XhoI fragment into a modified pBluescript vector digested with the same enzymes. Can anyone help me please? I have sequenced the linearized vector digested with both enzymes in order to confirm that the digestion is complete and that the cohesive ends are still in use. I've tried several vector:insert molar ratios, I've also tried the ligation reaction with and without dephosphorilated vector. Everything has been unsuccesful because all clones I obtain contain only religated vector. I don't understand how does the recircularization occurs but it happens. Control plates containing only vector (without insert) result only into one or two colonies. In contrast, 30-50 colonies are grown onto ligation plates. I don't know either how to explain this difference in number of cfu. When I screen all these colonies (either by PCR or by miniprep and restriction analysis) I only find vector. I don't know what else I can do. Any suggestion? I'm desperate!! Thank you very much for your help. Best wishes
Re: ligation problems
Posted by: P103 (IP Hidden, Unregistered user, )
Date: April 18, 2005 09:02AM
In my project at first, I found the same problem with you. My advisor told me that I should try to change the stock of E.coli competent cell. I don't understand why. But when I chenge competent cell the result show 100% ligated after restriction analysis. Hope you try and success like me
PS. Sorry for my poor English
Re: ligation problems
Posted by: nvergara (IP Hidden, Unregistered user, )
Date: April 18, 2005 09:14AM
Thank you very much for your suggestion.
I'm using chemically competent cells purchased from invitrogen (TOP 10 F'). When you suggest to change the stock do you mean the bacterial strain? Which cells did you use? Where they chemically competent or electrocompetent? Did you prepare yourself your competent cells? Sorry for all these questions and thank you very much again for your help.
Re: ligation problems
Posted by: P103 (IP Hidden, Unregistered user, )
Date: April 19, 2005 10:58PM
I use chemical competent cell prepared in our lab. At first the stock of E.coli DH5 ap. is too old or may contaminated. But in your case, I don't think that the quality comercial competent cell is your problem (Is it new? Because if it was kept for a long time, the quality of cell will be decreased. Please, check the production batch of the competent cell.)
I have been read about the incompatibility of competent cell and kind of vector (I mean that, the efficiency of transformation will be decreased). Good luck P103
Re: ligation problems
Posted by: fatboym (IP Hidden, Junior member, 20)
Date: April 20, 2005 01:10PM
I have just started working on transformation, so this may be just fool, but I wonder how have you cleaned your digested vector and insert?
Re: ligation problems
Posted by: nvergara (IP Hidden, Unregistered user, )
Date: April 21, 2005 08:09AM
I purified them from a agarose gel with QIAquick Gel Extraction kit (QIAGEN)
Re: ligation problems
Posted by: fatboym (IP Hidden, Junior member, 20)
Date: April 26, 2005 12:01PM
DO you dephosphorylate your vector. In theory this double digestion shouldn' t lead to any religation of the plasmid, but if the efficiency of the digestion is not 100% you may have problems
detection of transgene by pcr
Posted by: pcrgirl (IP Hidden, Unregistered user, )
Date: April 28, 2005 07:02AM
I am doing a pcr on a certain transgenic crop but my problem is that i'm getting nonspecific products. And my expected amplicon is very very faint. I already did a touchdown. I also increased the DNA in the reaction (100ng). And i think the components of my pcr cocktail is ok coz i'm getting a very strong band in my plasmid(as my postive control). I want to have a distinct and strong band to validate the presence of the transgene in the crop im working on, could you please help me? Thanks in advance.
Re: ligation problems
Posted by: LUI (IP Hidden, Unregistered user, )
Date: April 28, 2005 08:57AM
Cloning had been a large difficulty in my research before. So I hope sharing my experimence will be helpful for you.
Before l used gel purified DNA for ligation and then transformation, however, I found that the colonies on the plate contained vector only. But interestingly, when I change to use PCR purification kit to purify the enzyme digested insert/vector, after ligation and transformation, I could find my target plasmids. So in my opinion, I think you can try several things: 1. Use PCR purification method to purify your digested DNA 2. Ensure your competent cell is in a good condition, if not, prepare fresh competent cells following standard protocol. 3. Ensure the digestion of your vector/ insert is complete. Use sequencial cut instead of digesting the vector/insert with 2 enzyme simultaneuosly. Hope these suggestion can solve your problem!
Re: ligation problems
Posted by: devanarayanan (IP Hidden, Unregistered user, )
Date: April 30, 2005 11:28AM
Hai,
I am trying to clone 1.5kb pcr product by TA cloning method .For the last two months i am trying to clone it bu i am not able to do this .But when i am doing the same thing for soe other pcr products it is working fine.If anybody can give some suggestions it will be helpful thanks
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