IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/19

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Team Flavor

DTL of Mira/Brazz+StrepII into STOP+V0120

Digestion

Digestion Reactions
Stop v0120 1 Stop v0120 2
DNA 10 8
FD Buffer (10x) 2 2
diH2O 6 8
Xba1 1 1
EcoRI 1 1
Ligation Reactions
Mira N + Stop Mira C + Stop Brazz N + Stop Brazz C + Stop Stop only Control
diH2O 3 3 11 6 14
T4 DNA ligase buffer (10x) 2 3 2 2 2
DNA Insert 11 11 3 8 0
DNA Backbone 3 3 3 3 3
T4 DNA ligase 1 1 1 1 1


Confirm of valencene PCR (failed)

  • Re-do of Valencene PCR (failed)

Team Allergy

  • Got back sequencing results of GerS, BetS, PDK
    • All three samples of GerS and PDK sent in matched their corresponding sequences
  • Ran PCR of Friday's digested B21. The gel suggested a partial digest: we saw two bands of backbone where we should have seen only one:

  • Sending 6 samples of amiRNA (LTP.5), 5 samples of amiRNA (LTP), 6 samples of amiRNA (Bet)
  • Second digest of B21 appears to have been more successful:


Digestion Reactions
V0120
DNA ~2.1 ugrams
FD Buffer (10x) 2
diH2O 20-rest
Xba1 1
EcoRI 1
  • phosphatized backbone afterwards for 20 min after letting digestion run for an hour
  • concentrations
    • backbone: (5 copies each ~ 35 ng/uL)
    • Bet S/A:  ; Bet2S/A:  ; GerS/A:  ; LTPS/A:  ; PME:  ; Pal:
Ligation Reactions
BetS/A Bet2S/A LTPS/A GerS/GerA PmePal
diH2O 10-rest 10-rest 10-rest 10-rest 10-rest 10-rest
T4 DNA ligase buffer (10x) 1 1 1 1 1 1
DNA Insert 3 times excess 3 times excess 3 times excess 3 times excess 3 times excess 3 times excess
DNA Backbone ~50ng ~50ng ~50ng ~50ng ~50ng ~50ng
T4 DNA ligase 1 1 1 1 1 1
  • Ligated and transformed
    • allergen parts
    • Pme/pal


Team Fence

5xGal4 Promoter Annealing Product Ligation

  • Made a 1 in 1000 dilution of the annealing reaction


Nanodrop
ng/μL 260/280
5xGalpt 1/1000 dilution 17.6 2.25


For a ligation reaction, we want:

  • 50ng of backbone
  • A 1:3 ratio of backbone:insert
  • 3(ng of backbone / basepairs of backbone) = (ng of insert / basepairs of insert)

5xGalpt is 180 bases long, B21 backbone is 3.2kb. So we want 8.4375 ng of plasmid

To reach 8.4ng of 5xGalpt, made a 1 in 2000 dilution of the 5xGalpt Annealing product

Ligation Reaction:

  • 1μL 1/2000 5xGalpt
  • 2.5μL B21 Backbone
  • 6.5μL DH2O
  • 1μL Quick Ligase
  • 10μL 2x Quick Ligase Buffer

B21 Control Ligation:

  • 2.5μL B21 Backbone
  • 7.5μL DH2O
  • 1μL Quick Ligase
  • 10μL 2x Quick Ligase Buffer

Incubated at room temp for 15 mins, put on ice and immediately transformed.

Transformations of 5xGalpt, NLS.Serine anneal, and B21 Backbone control

  • Thawed 3 Turbo chemically competent cells on ice
  • Pippeted 1μL of the ligation or annealing reaction into its own tube of turbo cells
  • Chilled on ice for 30 mins
  • Heatshocked in 42°C water on heat block for 30 seconds
  • Chilled on ice for 2 mins
  • Added 170μL SOC medium to each tube, set to shake at 37°C for 15 mins
  • Streaked on LB + amp plates, and left in incubator overnight



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