User:Anthony Salvagno/Notebook/Research/2010/12/15/New Tweezer Construct: Designing the DNA

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How should I organize this page? I don't think I currently have a planning category. Should I make one? With that said I'm going to be building a new unzipping/stretching construct. We want to use a new plasmid that we can buy cheaply. Perhaps one with the same BstXI site as pRL574 so that we don't have to redesign the adapter (but I think I want to make that anyway because I want to add a few biotin more to the adapter to enhance microsphere attachment). I will start by finding a plasmid with that site and then design 2 or more PCR reactions (4.4kb reaction and 1.1kb reaction).

The Search for a Plasmid

The conditions:

  • cheap
  • contains exact BstXI cut site from pRL574

According to my Fermentas Reviewer tool (see the notebook homepage) the sequence I need to find in any plasmid I wish to build around is:

  • 5' - CCAACGAT | CTGG - 3' where the line is where BstXi snips. The overhang is CGAT meaning any compliment (like the one in our adapter) is 5' - ATCG.

Issues

This search is quite the struggle. Nothing seems to be able to locate the entire string that I listed above in any plasmid. I don't know if that is because there is some trick to the search that I'm not getting or if the plasmid databases I'm using is limited. I also don't know how to search for any restriction site that give me the 4 base overhang I need. This is complicated, but I feel like it shouldn't be.

Fixing the Issues

I sent an email to NEB and here was their response:

Dear Anthony, Thank you for contacting NEB Technical Support. Your question and answer are below. Your question: I would like to purchase a plasmid, but I need to be able to digest and achieve a specific 4 base overhang. Previously I was using pRL574 and digesting with BstXI to achieve the needed results, but I need a plasmid that I can purchase since I no longer have cloning capabilities. The recognition sequence that I was previously requiring was: 5'- CCAACGAT CTGG with the cut right after the first T. The overhang I need is 5' - CGAT and so any plasmid over 4kb could work. Do you have anything in stock that meets these requirements, or do you know of a tool that I can use to find a plasmid that suits this need? Any help would be greatly appreciated. Our answer: Unfortunately I do not know of any commercially available plasmid that meets your requirement. You could search plasmid sequences using the NEBcutter program; however BstXI sites are relatively uncommon, and your requirement for the specific 4 base sequence within the BstXI site means that on average only one in every 256 BstXI sites would have the correct sequence, so my guess is that it will be unlikely to find a suitable plasmid. Perhaps finding a way to have a prep of the pRL574 plasmid made for you would be the best solution? Unfortunately NEB does not offer such services.

I also asked Diego if he knew of anything that could help and while he didn't he was able to point out to me that there are two companies that make custom plasmids and we could get those. The companies are DNA 2.0 and IDTDNA. I emailed Dna 2.0 with essentially the same query as NEB only I also asked how much it would cost to have a custom vector made. Their response:

The easiest thing to do is ask us to synthesize a small fragment of DNA and clone it into our vector (pJ207) of size greater than 4kb (and lacks BstXI). We have a Minimum fee of $225, and a /bp rate of $0.75, so any fragment up to 300bp would cost the same. You can design this w/ a BstXI site, and anything else you like, and then you'll have a lifetime supply of this vector w/ the CGAT overhang.

Then I thought I could probably go through Alpha DNA and do something similar but definitely more time consuming. It also wouldn't be as reliable because I don't have the ability to blue select and transform vectors and so would just be relying on a bunch of crap. My suggestion was to double digest a plasmid like pBR322 (since we already have it) and then insert an oligo made by Alpha DNA that has the BstXI sequence in it with the matching overhangs for the double digest.

I do like the effortless purchase a custom plasmid option though, but money is tight. I'll have to see what Koch says.

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