IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/13

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Yeast Backbone




The yeast backbones containing the PIF3 and PHYB inserts was isolated and purified. The latter image shows where the appropriately sized bands were excised.

Nanodrop values of final product are:

PHYB 11ng/μL PIF3 20ng/μL

Arabidopsis Genomic DNA
A 150mg sample of arabidopsis leaves was attained from the Herbarium. Leaves were cleaned with de-ionized water and run through the ZR Plant/Seed DNA Miniprep kit from ZYMO RESEARCH according to the protocol describing extraction of DNA from plant tissue or seed. The extraction was only moderately successful and nanodrop values for the final product showed a 260/280 value of less than 1. The presence of contaminants in our arabidopsis genomic DNA sample may be the explanation for the unsuccessful PCR of arabidopsis promoters. Quantity of DNA in sample was approximately 20ng/μL.

Oligo Annealing NLS.Serine
Combined the following in an eppendorf: 3μL NLS.Serine.BB.Fwd 3μL NLS.Serine.BB.Rev 2μL 10x Annealing buffer 12μL DH2O
 * NLS.Serine.BB.Rev at 4.6nM, added 460μL DH2O
 * NLS.Serine.BB.Fwd at 2.4nM, added 240μL DH2O


 * placed tube in boiling water, let boil for 5 mins, then removed water from heat block and allowed the water with the tube in it to cool slowly on the bench.

Ligation in V24

 * Ligation of of Mira/Brazz+YFP+NOSt+STOP Construct into the V24 pETDUET vector for expression of proteins in IPTG inducible bacteria.


 * 5μL of Ligation reaction was added to 15μL TURBO cells. Transformation procedure was followed.

Team Allergy
Digestion


 * add phosphatase to backbone (15 min)


 * gel purify backbones LTPS: 8.7 ng/uL  Ger: 9.4 ng/uL
 * pcr purify digested pdk: PDK1 14.4 ng/uL; PDK2 22.4 ng/uL; PDK3 11.4 ng/uL; PDK4 15.3 ng/uL

Ligations

Ligations of PDK w/ V0120 (lacking YFP)

Ligations of PDK/Pal into V0120 + Sense Allergens (LTP & GER)


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