User:Lu Wang/Notebook/Team Allergy/2010/06/25

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Procedures

Today, we will take the PCR products from arabadopsis genomic DNA and transform the allergen panel inserts into E. col. Concurrently, we will create amiRNA constructs using PCR for allergens LTP and Betv1.1, as well as control protein GFP.

Unlike our work for our allergen panel, amiRNA does NOT require sense and antisense parts of the original gDNA. The reverse complement sequences that are inserted into RS300 is inserted using oligos, and our insertions are only ~20bps in length. We used Web MicroRNA Designer to optimize the sequence of our insertion oligos, which will be the insertion sequence followed by 20bps of the RS300 sequence. PCR reactions will incorporate these additional sequences into the RS300, inducing mutagenesis.

For hpRNA

  1. Digest arabadopsis allergen panel with Xba1 and Pst1
  2. Purify digested DNA
  3. Digest vector V0120
  4. Purify Vector
  5. Ligate Vector and Inserts
  6. Transform

For amiRNA

  1. Set up 3 step PCR using RS300 as template and primers I,II,III, and IV from website and primers A and B that we designed
  2. Stitching PCR reactions: both 3-in-1 and 2-in-1 reactions

Results

For hpRNA

Requesting table of concentrations of purified DNA after digestion.

Transformations worked for all

For amiRNA During PCR, ran LTP and GFP with both 1μL and 0.5μL of RS300 template because the concentrations of the template was sufficiently high. Plasmid PCRs require only 1~10ng of DNA (tiny compared to the 100~250ng necessary for genomic DNA PCR).

Gel electrophoresis of the stitching PCR showed that all LTP and GFP genes were successfully ligated in the 3-in-1 stitching PCR. The construct should be 450bps in length. We will redo the three step PCR tomorrow to try and see if we can correctly create Betv1.1 constructs.


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