User talk:Anthony Salvagno/Notebook/Research/2009/03/11

From OpenWetWare

Jump to: navigation, search
  1. 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?
  2. 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.
  3. 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.
  4. 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:

  1. I think it means we need a higher ratio of vector to plasmid
  2. 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.
  3. 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.
Personal tools