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Talk:DNA ligation: Difference between revisions
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→Insert to Vector ratios
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**Not suggestions but questions. I apparently have a setup that is hard to insert. V6.3kb:I2.2kb and have only the slightest evidence that V:I has formed, let alone circularized. I am noticing in the literature that optimal ligations are between 4 and 16'C (O/N) and that ligation ratios are between 1:1 for 'cip' treated vectors to 5:1 for blunt end ligations. Lots of contradictions, also people recommend vector concentrations of 10 fMole and insert of 30 fmole, but then say don't add to much DNA, no more than 20 ng per. Some people recommend preheating the vector and insert after mixing 37'C, 50'C, 65'C (drive iff residual ethanol or unstick the ends). For the first time user it is helpful to know what is most important (fmoles/ul or ngs/ul), what to do and when to do it. | **Not suggestions but questions. I apparently have a setup that is hard to insert. V6.3kb:I2.2kb and have only the slightest evidence that V:I has formed, let alone circularized. I am noticing in the literature that optimal ligations are between 4 and 16'C (O/N) and that ligation ratios are between 1:1 for 'cip' treated vectors to 5:1 for blunt end ligations. Lots of contradictions, also people recommend vector concentrations of 10 fMole and insert of 30 fmole, but then say don't add to much DNA, no more than 20 ng per. Some people recommend preheating the vector and insert after mixing 37'C, 50'C, 65'C (drive iff residual ethanol or unstick the ends). For the first time user it is helpful to know what is most important (fmoles/ul or ngs/ul), what to do and when to do it. | ||
***'''[[User:Reshma P. Shetty|Reshma]] 14:21, 7 September 2007 (EDT)''': Ahh ok. How is your insert generated? By PCR followed by digestion or by digesting it from another vector? If it is digested from another vector, does the insert vector and the ligation vector have the same resistance marker? Have you've tried this cloning once already and it failed? If so, can you describe your conditions? | ***'''[[User:Reshma P. Shetty|Reshma]] 14:21, 7 September 2007 (EDT)''': Ahh ok. How is your insert generated? By PCR followed by digestion or by digesting it from another vector? If it is digested from another vector, does the insert vector and the ligation vector have the same resistance marker? Have you've tried this cloning once already and it failed? If so, can you describe your conditions? | ||
****'''[[User:Philip R. Deitiker|Phil D]] 12:25, 9 September 2007 (CDT)'''There have been several things I have tried to clone, the vector can be cut and recloses with efficiency, which can be verified by cip treating it. The ligation of smaller products without transformation was successful, amplified by PCR. Several vector/insert products have been attempted, no success. | ****'''[[User:Philip R. Deitiker|Phil D]] 12:25, 9 September 2007 (CDT)'''There have been several things I have tried to clone, the vector can be cut and recloses with efficiency, which can be verified by cip treating it. The ligation of smaller products without transformation was successful, amplified by PCR. Several vector/insert products have been attempted, no success. | ||
Since lower temperature works better I will repeat, at 10'C for 24 hours and see if I can improve amount of product. It may be true that more insert forces more vector-insert product, this is the first time I have seen a ratio of 6:1 proposed. Has anyone had success using less PEG, might this improve the reactivity of vector ends? | :::So I have gone back to basic principles. The vector was EcoR1 digested and Cipped, heat killed the cip w/EDTA then added more magnesium and BamH1 digested, this blocks the 5' end of the linearized vector from closing or duplexing. The PCR product was cut with Bgl II (exension of 8 nt) but not EcoR1. In the latest attempt at ligation I am following a protocol 0.2 units of Enzyme 10 fM of vector, 30 fm Insert in 30 ul overnight at 14'C. This latest ligation was semi-successful. Majority of insert was in dimers and some vector-insert was barely (required trans illuminator box to see) visible, no visible vector-vector dimers. | ||
:::Since lower temperature works better I will repeat, at 10'C for 24 hours and see if I can improve amount of product. It may be true that more insert forces more vector-insert product, this is the first time I have seen a ratio of 6:1 proposed. Has anyone had success using less PEG, might this improve the reactivity of vector ends? | |||
