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

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Team Fence

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

  • NLS.Serine.BB.Rev at 4.6nM, added 460μL DH2O
  • NLS.Serine.BB.Fwd at 2.4nM, added 240μL DH2O

Combined the following in an eppendorf: 

 3μL NLS.Serine.BB.Fwd
 3μL NLS.Serine.BB.Rev
 2μL 10x Annealing buffer
 12μL 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.

Team Flavor

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.
Ligation Reactions
Mira N + V24 Mira C + V24 Brazz N + V24 Brazz C + V24 Control
diH2O 7 1 6 4 12
T4 DNA ligase buffer (10x) 2 3 2 2 2
DNA Insert 5 20 6 8 0
DNA Backbone 5 5 5 5 5
T4 DNA ligase 1 1 1 1 1
  • 5μL of Ligation reaction was added to 15μL TURBO cells. Transformation procedure was followed.

Team Allergy

Digestion

Digestion Reactions for Backbones (plasmids containing sense)
pLTPS1 pLTPS2 pLTPS3 GerS1
DNA 10 8 7 10
FD Buffer (10x) 2 2 2 2
diH2O 6 8 9 6
Spe 1 1 1 1
Pst 1 1 1 1
  • add phosphatase to backbone (15 min)
Digestion Reactions for Inserts
Pal2 PDK
DNA (~ 1 ug) 9
FD Buffer (10x) 2 2
diH2O 7
Xba1 1 1
Pst1 1 1
  • 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)

Ligation Reactions
Pdk1 Pdk2 Pdk3 Pdk4
DNA Insert 2.36 2.98 1.52 2.22
T4 DNA ligase buffer (10x) 2 2 2 2
diH2O 12.64 12.02 13.48 12.78
T4 DNA ligase 1 1 1 1
DNA Backbone (~50ng) 2uL 2uL 2uL 2uL


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

Ligation Reactions 3:1 excess
PDK1 PDK2 PDK 3 PDK 4
DNA Insert (~36ng/ ~32 ng)) 2.51; 2.27 1.62;1.46 3.18; 2.86 2.37;2.13
T4 DNA ligase buffer (10x) 2 2 2 2
diH2O 8.49; 9.73 9.38; 10.54 7.82;9.14 8.63;9.87
T4 DNA ligase 1 1 1 1
DNA Backbone (~50ng/(~47ng) LTP1S 6; GerS 5 LTP1S 6; GerS 5 LTP1S 6; GerS 5 LTP1S 6; GerS 5



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