User:Lu Wang/Notebook/Team Allergy/2010/07/01

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Procedures

Today, we are going to make constructs for amiRNA in V0120 without the death gene (B21, which contains YFP). We will also try to isolate introns from arabadopsis gDNA and redo ligations of allergen panel into V0120.

  1. Ligation for amiRNA construct and V0120
  2. PCR PME and PAL from arabadopsis gDNA
  3. Ligation of allergen panel into V0120

Results

  • 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 10 ng of insert (ratio of insert to vector ~.09375



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