Team:Alberta/Notebook/Assembly Method

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Revision as of 19:19, 22 July 2010

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).

26-05-2010

Designed new anchors, ordered oligos. Designed new anchors with larger polyA tail (and without hairpins, or self dimerization).

27-05-2010

No binding capacity specified in the manual for the beads in terms of mols or mass of DNA or beads. Binding capacity is given in terms of cells (because the beads are meant for mRNA isolation from cells). Will have to quantify binding capacity later.

Derivation of mole matching equation: ssDNA is ~330 grams/(mol*bp) moles = [concentration X volume] / [(330grams/mol X bp) X length] moles1 = moles2 c1v1/[(330g/mol X bp) X l1] = c2v2/[(330g/mol X bp) X l2] v1 = v2(c2 X l1/ c1 X l2)

Ordered new primers for anchor: GCG CGC CCG GTC TCA TGG GTC ACC CTC CC GGG AGG GTG ACC CAT GAG ACC GGC GCG C[A]12

07-06-2010

Made Wash Buffer, Elution Buffer and Low Salt Buffer for the mRNA Isolation Kit.

08-06-2010

Produce a modified protocol for Anchor binding to cellulose beads.

Protocol Preparation: Allow everything to come to room temperature. Spin 2000 to 5000g of beads in microcentrifuge for 10 seconds. Remove supernatant without disturbing the beads. Add 200:u;l of Wash Buffer to beads and agitate. Centrifuge for 10 seconds and remove supernatant. Prewarm Elution Buffer in a 70 degrees Celsius water bath.

Isolation Procedure: Apply DNA Anchor to cellulose beads, agitate and let it stand at room temperature for 5 minutes. Microcentrifuge for 10 seconds. Pipette supernatant back into original microcentrifuge. [Add 4:u;l Wash Buffer to beads and agitate to resuspend. Transfer beads and wash buffer to column reservoir of spin column. Let it stand at room temperature for 2 minutes while agitating. Microcentrifuge for 10 seconds.] X3 Add 400:u;l Low Salt Buffer, resuspend by agitation for 2 seconds and microcentrifuge for 10 seconds. Transfer and place spin column reservoir in a clean microcentrifuge tube. [Add 200:u;l pre-warmed Elution Buffer to column reservoir. Agitate to resuspend beads and let it stand for 2 minutes. Microcentrifuge for 10 seconds] X2 Place eluent on ice.

09-06-2010

<p>Calculated the mass of beads to determine the range of mg RNA could be isolated.

Calculated the minimal and maximal rpm for 2000 to 5000g of beads using the following formula: a = 4(pi)^2r(rpm)^2 / 60^2 So, when centrifuging the beads, the rpm must stay between 4700 to 7400 rpm. In the modified mRNA procedure, the centrifuge is set to 5500 rpm.