User:Lu Wang/Notebook/Team Allergy/2010/06/21

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Faculty Presentation 2
Team Vector, Team Flavor, Team Allergy, and Team Fence presented their progress from the previous week of June 14th to June 18th. A copy of the presentation can be found here.

Some questions and comments that came from the faculty for the various teams. Obviously, these are incomplete, but I will request input from members of the other team.

Team Vector - how to insert oligos into their vectors? - do the biobricks code for any unusual proteins or cause any change/damage to the plants?

Team Flavor

Team Allergy - what happens when we remove the allergen proteins? - what were the results from other similar experiements?

Team Fence

Our next faculty presentation will be in one week on June 28th.

Strawberry hpRNA Interference
The gene coding for Fra a 1 has a 150bp exon (we're going to call it exon fra1), followed by a 150bp intron (intron fra1) and 300 exon (exon fra2). The next time we isolate mRNA from strawberries, we are going to isolate mRNA for exon fra2 only. Including an intron in the sense/antisense of the hpRNA may cause the plant to have multiple targets for interference, and we only want to shut down genes coding for fra a 1. Therefore, we will only use one exon. We will use exon fra 2 because we need a hpRNA at least 1000bps long, and exon fra1 may be too short.

Procedures
Today, the objective is to obtain PCR products of Arabadopsis allergens LTP 1, Ger 3, and Bet v1.

1. Obtain wildtype Arabadopsis genomic DNA 2. PCR for each LTP 1, Ger 3, and Bet v1

Results
1. Obtain wildtype Arabadopsis genomic DNA

Obtained from the Harvard Herbaria. Nanodrop yields 15.7 ng/μL.

2. PCR for each LTP 1, Ger 3, and Bet v1

Only LTP sense, Ger 3 sense, and Ger 3 antisense were successful. Concentrations were 2.6, 7.8, and 7.9 ng/μL, respectively.

The other genes were not amplified. There are many reasons for why LTP antisense and Bet v1 did not amplify, so we tried to eliminate some potential problems. After checking the primers against the arabadopsis genome, the primers seem correct. Annealing temperatures also fall within the appropriate range.

Other possible problems could be contamination, improper pH, insufficient DNA, primer design beyond sequencing (unlikely), or incorrect primers in a reaction (unlikely). We will obtain more arabadopsis genomic DNA and try PCR again.


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