User:Lu Wang/Notebook/Team Allergy/2010/08/02

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We have correct hpRNA for allergens LTP, Bet v2, and Ger. Bet v1 was not successful because a band did not show on a gel after the Sense and Intron ligation. The goal now is to get the working hair pins into plants. Eventually, we'll go back and redo Bet v1 + PAL and all the PDK ligations to see if it was just bad luck we had before, if we did something wrong, or if those parts don't work period.

Today, we are going to take our functional hairpins and put them into vectors V9 and V10, compatible with both E. coli and agrobacteria. We will grow up V9 and V10 containing our hpRNA, extract the plasmids, and introduce them into agrobacteria. V9 and V10 are made with expression promotors. The inserts for V9 and V10 will be cut with the same restriction enzymes.

  1. Digestion of V9 and V10
  2. Phosphorylation of V9 and V10
  3. Digestion of Allergen hpRNA
  4. Nanodrop
  5. Ligations of hpRNA and V9 or V10
  6. Transformation into E. coli


Digestion of V9 and V10

We digested 1000ng of each vector using the fast fermentas digest protocol. Digested 2μL (1000ng) of each V9 and V10 with 2μL 10X FD buffer, 1μL of each Pst and Eco restriction enzymes, and 14μL distilled water. Incubate for 30 minutes at 37°C.

Phosphorylation of V9 and V10

Added 1μL of phosphorylase after digestion, incubate at 37°C for 20 minutes.

Digestion of Allergen hpRNA

We digested approximately 100ng of each of the samples of hpRNA. We used the fast fermentas digest protocol. Digestions were done with 20 μL reactions. A few samples did not contain 100ng after 100ng was sent for sequencing, so we digested all of those samples that remained.