User:Ramalldf/Notebook/Research/2009/03/13

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3/13/09

  • I have the complete set of notes in my notebook, but this is just to be able to share with you.

AscI Restriction Digest of TOPO Clones

  • I digested with AscI just like before, and ran a gel using both new and old Kb + ladders.
  • Here is the content of each lane (10 ul of between 70-200 ng/ul for each digested sample):
This is the digest that Diego did today. It looks like we cloned something in there! So the large band of the double digested samples is about 4 kb which should be about what we would expect if we had all of our inserts in there (2.6 kb for vector + 2 kb for SLH) Looking at it again, I'm thinking that it might've come out clearer if I took the gel off of the tray when I took the picture.
    • Top (wt):
      • L1: New kb+ ladder
      • L2:Uncut WT plasmid (sample 6)
      • L3:Empty
      • L4:Control 1
      • L5:Empty
      • L6:Digest sample 1
      • L7:Empty
      • L8:Digest sample 3
      • L9:Empty
      • L10:Digest sample 6
      • L11:Empty
      • L12:Digest sample 9
      • L13:Empty
      • L14:Old kb+ ladder
    • Bottom(L99 Mut):
      • L1: New kb+ ladder
      • L2:Uncut Mut plasmid (sample 6)
      • L3:Empty
      • L4:Digest sample 1
      • L5:Empty
      • L6:Digest sample 3
      • L7:Empty
      • L8:Digest sample 6
      • L9:Empty
      • L10:Digest sample 9
      • L11:Empty
      • L12:Control 2
      • L13:Empty
      • L14:Old kb+ ladder
  • By the controls, I mean the set of rxns where we did the polishing step without any of our DNA in there that we later cloned, transformed, miniprep-ed and now digested.
  • Let me know what you think! (email if you don't have an openwetware account).

Interpretation

  • Ok, after looking at it again I think that it makes a little more sense. The control samples have bands on there because of the TOPO vector and only have one band because they were linearized by AscI (which had one AscI site in and of itself).
  • The samples that have the double bands on there (ie. all of the non-control digests in the bottom gel) prove that there's an insert in there because:
    • There's two bands (could only be a result of two restriction sites on the plasmid, one from vector and one from Halo insert).
    • The larger band is larger in size than the the linearized bands for any samples that did not have the vector (ie. weren't double digested).


Conclusion

  • Of our colonies that we miniprepped, only sample #6 of the WT samples had the insert (Since the band heavy band in the double digested sample 6 has a weight between 4 and 5 kb).
  • However, all of our mutant samples seem to have the insert.
  • One thing that confuses me: Our heavy band on the WT digested sample 6 matches our undigested sample 6 middle band (linearized), which I would expect to see if there was incomplete digestion, but I'd still expect to see another band that would be smaller than that (ie. plasmid minus the small band).
  • Another confusing thing: I thought that Alex said that our small band should be about the size of Halo (about 700 bp), but it looks to be more about 850 bp in size. WTF?