"

From 2010.igem.org

25-05-2010

Discussed an assembly method. Will start with a polyA tail Anchor oligo which will connect to a polyT tail which is bound to the bead. The anchor will consist of a primer region, a BsaI cut site, a buffer region, and finally, an A prime or B prime end. A byte with an appropriate end can be added to this anchor, and another byte can be added onto that, etc... The last thing to be added to the construct is a Terminator byte consisting of a buffer region, bsaI cut site, primer region and a polyT tail. This second primer sequence must be as uncomplimentary as possible to the anchor's primer site so as to minimize primers binding to each other when PCRing the assembled construct. The primers melting point must be around 65 C. After the construct is assembled on the bead, it is heated off and the polyA tail of the anchor binds to the polyT tail of the terminator and a plasmid is born.

We will purchase NEB oligo dT magnetic and cellulose beads which have a polyT tail (5' to 3') 25 nucleotides long. They are meant for mRNA isolation, but should work fine for our purposes. Used the IDT Analyzer software to determine what length of polyA tail is required for our anchor to have a melting temperature of 30 degrees Celcius (decided to change this melting temp later). This melting temp is sensitive to Na+ and Mg++ concentrations, so referred to ligase buffer and elution buffer for these concentrations.

The BsaI A end should look like this:

Designed an anchor with a polyA10 tail with a primer (Tm=65.1 C) region and the BsaI cut site with an A' end (ACCC).

style="color:#1b2c8a;background-color:#FFFF33;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"

!align="center"|Notebook !align="center"|Building Parts !align="center"|Testing Parts !align="center"|Assembly Method !align="center"|Competent Cells !align="center"|Plates !align="center"|Kit Manual !align="center"|Software |}