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Talk:DNA ligation: Difference between revisions
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#Crowe-NAR-1991 pmid=2011503 | #Crowe-NAR-1991 pmid=2011503 | ||
</biblio> | </biblio> | ||
==Ligation troubbles== | |||
[[Image:BHOG080507_gel1.jpg|thumb|500px|1% agarose gel, 0.5 mg/mL EtBr<br>0.5x TBE 0.5hrs@130V]] | |||
*'''[[User:Bjorn Hogberg|Bjorn Hogberg]] 14:13, 7 May 2008 (EDT)''':Hi, I'm having some real difficulties with my ligations (and/or transformations). The goal is to insert a 1.4kB PCR amplified insert into a 7.2kB M13mp18 vector cut with BamHI and HindIII. I have tried a lot of different strategies including: gel purification of insert vs. PEG purification of insert, room temp ligation vs. 16°C and 4°C ligations, ligation overnight vs. just an hour, Antrarctica Phosphatase treatment of vector vs. no treatment and a bunch of other stuff like varying ratios vector:insert. This gel is results from a 4°C overnight ligation (T4 DNA ligase from NEB) on Ant.Phosph. treated vector, I use 50ng of vector in a 20µl volume. It seems like the insert is ligating to itself a lot but never to the vector. Any suggestions, I'm desperate. | |||
I can also say that I have tried transforming on several occations. Once I thought it was alright because I got very few plaques on a negative control. And a lot of plaques on a 1:10 ligation plate, but alas, no insert was found after screening. | |||
