IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/01

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

Team Flavour

  • We made glycerol stocks from our Miraculin & v0120 and Brazzein & v0120. 333 μL cultures with 666μL glycerol and placed in -80°C freezer.
  • We miniprepped our Miraculin and v0120 cultures and our Brazzein and v0120 cultures. Miniprep done per Qiagen protocol.


ODs

Sample OD
Miraculin and v0120 #1 171.3ng/μL
Miraculin and v0120 #2 202.0ng/μL
Miraculin and v0120 #3 106.4ng/μL
Miraculin and v0120 #4 115.6ng/μL
Miraculin and v0120 #5 40.5ng/μL
Brazzein and v0120 #1 41.7ng/μL
Brazzein and v0120 #2 42.8ng/μL
Brazzein and v0120 #3 38.8ng/μL
Brazzein and v0120 #4 0ng/μL
Brazzein and v0120 #5 N/A


Valencia orange surgery (part two)

  • RNA extraction per Qiagen RNAeasy protocol.
    • We made sure to apply RNAzap (RNAase-free) spray to all surfaces and instruments.


ODs

Sample OD
RNA Flavedo extraction #1 93.1ng/μL
RNA Flavedo extraction #2 28.7ng/μL

Team Fence

B21 Backbone Gel Extraction

  • followed QIAquick gel extraction handbook except melting temperature was 42°C instead of 50°C
  • 4 gels to begin with, highest nanodrop value before consolidation = 1.4ng/μL
  • The 4 tubes were consolidated into 2 and placed in the vacufuge for ≈30 minutes to increase DNA concentration
  • The 2 tubes were consolidated into 1 for a final concentration of 18.6ng/μL

PMT1002 Digestion

  • 2 reactions each containing the following:
    • 1μL EcoRI
    • 1μL HindIII
    • 2μL 10X digestion buffer
    • 1/2 ng of sample ≈7μL
    • 9μL dH2O

only one band was visible in the gel for each reaction. Julia forgot to put the ladder in so it was not possible to precisely determine the single band size, but judging from the position of the loading dye it seemed that the plasmid had only been cut once.

Ligation

Ligation performed on Barstar, NLS, and LacIN

  • 3 times as much insert as vector per reaction, length of insert taken into consideration.
    • Barstar: 0.4 kb = 18.5ng
    • NLS: 0.06 kb = 3ng
    • LacIN: 1.1 kb = 52 ng

Ligation Recipe:

  • 10μL insert + backbone
  • 10μL ligation buffer (vortexed thoroughly)
  • 1μL ligase, mix gently

let stand for 15 minutes at room temperature, then put on ice use for 4μL of the product for transformation and freeze the rest

LacIN

  • 3μL B21 vector
  • 7μL LacIN insert

NLS

  • 3μL B21 vector
  • 1μL NLS insert
  • 6μL dH20

Barstar

  • 3μL B21 vector
  • 1.7μL Barstar
  • 5.3μL dH20

Transformation from ligation

  • thawed 4 chemically competent TOP10 cell tubes on ice
  • added 4μL of each ligase rxn to each incubated on ice for 30 minutes
  • heat shock at 42°C for 45 seconds
  • put on ice for 2 minutes
  • added 170μL SOC medium to each tube set to shake at 37°C for 30 minutes
  • pippetted each tube onto its own LB+amp plate, streak with beads
  • incubate at 37°C overnight