IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/01: Difference between revisions

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*gel: lane 1:ladder; lane 2: Pal3; lane 3: Pme 2; lane 4: ladder
*gel: lane 1:ladder; lane 2: Pal3; lane 3: Pme 2; lane 4: ladder


[[Image:PAL2_PME3.jpg| Pal 2 and Pme 3 introns|250 px]]
*'''Sent amiRNA Bet for sequencing'''
*'''Sent amiRNA Bet for sequencing'''


Revision as of 19:40, 1 July 2010

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Team Allergy

  • Ligations for full amiRNA and V0120 (w/o death gene)

note: .5 denotes multistep pcr reaction from yesterday in which .5 uL of part 3 of gfp/ltp were use in one of the giant assemblies of parts 1,2,3 w/ primers A&B

Ligation Reactions
Bet GFP LTP LTP(.5) GFP(.5)
DNA Insert (after 1:10 dilutions) 3 4.5 3 4.71 4.1
T4 DNA ligase buffer (10x) 2 2 2 2 2
diH2O 11.5 10 11.5 9.79 10.4
T4 DNA ligase 1 1 1 1 1
DNA Backbone 2.5 2.5 2.5 2.5 2.5
    • Used 50 ng of backbone and 150 ng of insert (ratio of insert to vector ~.14
    • Did 1:10 dilutions for the digested inserts


  • PCR of PME3 and PAL2
    • saw no bands from last night's pcr so we re ran it

Annealing Temp: 69C Extension Time: 30 sec

2 reactions (PME & PAL)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
Arabadopsis Genomic DNA 1
Fwd/Rev Primer .5 uM each (1uL 50x)
Water 35.5

Concentrations: PME: 250 ng/uL PAL: 134 ng/uL

  • gel: lane 1:ladder; lane 2: Pal3; lane 3: Pme 2; lane 4: ladder

Pal 2 and Pme 3 introns

  • Sent amiRNA Bet for sequencing
  • Ligation of Antisense/sense allergens into V0120 (lacking death gene)
Ligation Reactions
BetS BetA LTPS LTPA GerS GerA Bet 1.2S Bet 1.2 A Negative Control
DNA Insert (after 1:10 dilutions) ~ 3 times excess 3 3 3 3 3 3 3 3 0
T4 DNA ligase buffer (10x) 2 2 2 2 2 2 2 2 2
diH2O 12 12 12 12 12 12 12 12 15
T4 DNA ligase 1 1 1 1 1 1 1 1 1
DNA Backbone 2 2 2 2 2 2 2 2 2
    • Used 34 ng of backbone and 102 ng of insert (ratio of insert to vector ~.09375



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