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Talk:DNA ligation: Difference between revisions
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*'''[[User:Reshma P. Shetty|Reshma]] 19:50, 6 September 2007 (EDT)''': Good catch. I think this may reflect the fact that depending on exactly what you are ligating, the optimal insert:vector ratio can differ so different people advocate different ratio's. Hence the note in bold regarding the insert:vector ratio. If you have suggestions on ligating long inserts, please feel free to add it to the Notes section. | *'''[[User:Reshma P. Shetty|Reshma]] 19:50, 6 September 2007 (EDT)''': Good catch. I think this may reflect the fact that depending on exactly what you are ligating, the optimal insert:vector ratio can differ so different people advocate different ratio's. Hence the note in bold regarding the insert:vector ratio. If you have suggestions on ligating long inserts, please feel free to add it to the Notes section. | ||
**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? | |||
