User:Anthony Salvagno/Notebook/Research/2010/12/17/Custom Plasmid for Unzipping/Stretching Construct
I will get a 5kb vector from DNA 2.0 and have them add the sequence:
- 5' - CCAACGATCTGG
which produces the overhang CGAT, which is complementary to our adapter duplex. I'm allowed for anything up to 300bp (because it is the same price) so I'm pondering if I should add more too it or not. Right now I'm going with no and am requesting a quote. If I did add more what would it be?
For shits and giggles I'm going to add a SapI recognition site in before the BstXI site, This sequence would be:
- 5' - GCTCTTCCAGCTC
Because of the nature SapI it will leave a 5' overhang of AGC (cutting right after the CC above) which can be ligated onto the adapter duplex for unzipping.
Koch also wants me to add the high-affinity nucleosome binding site. I'm not sure this is what I need but:
- 5' - ctggagata cccggtgcta aggccgctt aattggtcgtagcaagctctagcaccgcttaa acgcacgtacgcgctgtcta ccgcgttttaaccgccaata ggattactta ctagtctct aggcacgtgta agatatatacatcctgt
I got this sequence from section 4.1 from here.
Once I get a quote I will ask for the vector sequence so that I can plan the PCR reaction.
The vector sequence can be found here:
/BstXI Custom Plasmid Sequence - this thing should have a cool name. How about Plasmid Destroyer 8003? I could name it Vector Sigma in honor of transformers, but that is gay. I know, IheartPlasmid... yea!
The plasmid I want is 5kb. I want to design 2 PCR reactions, but 1 at first. The first one will be 4kb with the forward primer containing the dig molecule and the reverse primer will contain a biotin and will anneal about 10 bp from the BstXI site. This way the site can be cleaved with BstXI if I want to do unzipping constructs or the molecule can be stretched if I want to go straight into tethering. I think this also allows for the attempt at ligation on a slide or restriction on a slide which could be a cool experiment.
I want the reverse primer close to the cut site so that when I use the PCR cleanup kit, the digested piece will go right through the membrane without doing a gel slice. I also want the PCR to yield a 4kb fragment so that tethering works with both big and small beads. With the short fragment, tethering isn't all that efficient when dealing with big beads.