User talk:Anthony Salvagno/Notebook/Research/2009/03/11
- Steve Koch 17:26, 11 March 2009 (EDT): Bummer that it didn't work! Does the fact that you got a single colony when you put in more vector (relative to gDNA in the ligation reaction) mean anything?
- Steve Koch 17:30, 11 March 2009 (EDT):About the small fragments theory: didn't you gel extract after XhoI digestion of the gDNA to select for medium-sized fragments? Since you're treating the pRS plasimd with CIAP, can't you go ahead and put in a 10:1 or whatever high ratio of plasmid to insert?
- Steve Koch 17:43, 11 March 2009 (EDT): Also, based on Larry's work, there really aren't that many small fragments produced by XhoI digestion of yeast genomic DNA. So I don't know whether it's a worry and whether the gel extraction step is necessary. If you do want to filter out small pieces, I think a spin column based on size-exclusion would work much better. I like the MicroSpin S-400 HR columns from GE Healthcare (previously Amersham). This link probably won't work because life science supply sites are awful: http://www1.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=975946&moduleid=166250&zone= It is ridiculously easy to use, and it should greatly increase the proportion of larger fragments relative to smaller ones, and have a really high yield for large fragments.
- Steve Koch 17:43, 11 March 2009 (EDT): As for concatamers of genomic DNA, that can be prevented by using CIAP (or I guess you call it CIP) on the genomic digestion. Of course, then you can't do the same for the plasmid digestion, so you'd get a bunch of recircularized plasmid. That's OK if you have a blue/white selection (to let you know whether the insert made it into the plasmid). This sounds more appealing to me than what you're doing now.
- Steve Koch 17:47, 11 March 2009 (EDT): I found a paper where they use shotgun cloning (of blunt end fragments from ChIP actually) and use a kit from Invitrogen. I don't know whether a kit would be helpful here. But I do note that they use a strategy of using CIP on the genomic fragements, and thus their plasmid, OneShot TOP10, must have some kind of blue/white selection.
Anthony Salvagno 18:08, 11 March 2009 (EDT):Answers to your questions:
- I think it means we need a higher ratio of vector to plasmid
- We did not gel extract the gDNA, just the plasmid. Kelly figured we should go with the whole lot for more randomness. I think next time I will say lets just just out some piece because we can't really get any less random, just selection size is less.
- Steve Koch:Check out those S-400 sephacryl spin columns I mentioned. They are really easy to use. Also great for getting rid of enzymes.
- So we are doing CIP (CIAP) on the plasmid. Do you think its better to do it for the gDNA instead?
- Steve Koch:Yeah, I do, because concatamerization wouldn't happen. But it requires some kind of selection (blue / white?) for clones that didn't get an insert. But I could be wrong.