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Talk:DNA ligation: Difference between revisions
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:::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. | :::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? | :::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? | ||
*'''[[User:Reshma P. Shetty|Reshma]] 11:28, 11 September 2007 (EDT)''': Hmm. I'm afraid I do my clonings pretty differently from what you've outlined so it is hard for me to debug. It sounds as if it is not necessarily your ligation that is failing. It could be a combination of low efficiency cutting of your insert with low efficiency ligation equals very little product that is correctly ligated. Some ideas ... | |||
#Regarding PEG, [http://www.neb.com/nebecomm/products/faqproductM0202.asp#339 NEB says that overnight ligations can inhibit transformation efficiency because of the PEG] ... so can heat killing ligation reactions (heat treated PEG inhibits transformation efficiency). So it sounds like PEG impacts transformation but not necessarily ligation efficiency. | |||
#One place where I have had problems in the past is getting efficient digestion of PCR products. Supposedly the theory is that polymerase can hang around attached to the DNA and prevent ligation (or fill in digested ends). My solution is to do a [[Knight:TOPO TA cloning|TOPO cloning of the PCR product]], screen by colony PCR and subsequent sequencing of miniprepped DNA. Then digest the miniprepped DNA for cloning into the vector of my choice. It adds an extra cloning step but its worked for me where direct cutting of a PCR product and cloning failed. Alternatively, you could treat the PCR with proteinase K ... see reference below. I have not tried this approach but others in my lab have. However, based on the fact that your ligation of smaller products seems to be working and your latest ligation with BglII cut PCR product produced insert dimers, perhaps this is not the problem. (Although perhaps the EcoRI digest of the PCR product is less efficient or getting both BglII and EcoRI to cut your PCR product is less efficient?) | |||
#The volume of the restriction digest can sometimes impact digest efficiency (according to my advisor ... he heard this from NEB). I do my restriction digests in a 50μL volume (small volumes ... say 20μL can be problematic apparently). | |||
#I prefer [[Phosphatase treatment of linearized vector|using antarctic phosphatase]] rather than CIP. The antarctic phosphatase can be killed with heat as opposed to the EDTA you're using for CIP. Could the added EDTA be impacting the ligation? I'm not sure. | |||
#I tend to do my ligations with 50ng vector and ~3-fold molar excess of insert. I am almost always doing 3-way ligations which is why I use so much DNA. I think you should be able to get away with less but these amounts work reliably for me. | |||
I am not sure that any of these ideas are very helpful to you given the controls that you've done. If I come up with any more insights, I'll let you know. | |||
<biblio> | |||
#Crowe-NAR-1991 pmid=2011503 | |||
</biblio> | |||
