IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/05

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dspB Track

  • Transformants from last day (Aug 04)
Transformant Results
Plate # of colonies
1no colonies
2no colonies
31 colony
4no colonies
5no colonies
6no colonies

Colony PCR

Protocol: See common protocol
Changes: use Phusion pol

  • Colony PCR plates 2 + water control (W)
PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x210
10mM dNTP2x24
sdH2O10.55x221.10
Phusion polymerase0.2x20.4
MgCl22x24
DMSO - 5%1.25x22.5
Total25
  • To 3 and water, add 2uL of fw + rev primer each, total 4uL of primers
  • To 3 and water, add G1004 (77.12C) & G1005 (75.38C) [Tm calculated using Finnzymes.com calculator)
  • Pick colony from respective plates to respective tubes

PCR Cycles:

  • 98C @ 3min
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1318
End: 1421

Gel verification for colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 1.2% agarose gel
  • Machine conditions: 0.5x TBE buffer, 100V, 45min

Gel orientation:

Gel orientation
3100bp ladderW (control)

Results:

Restriction Digest

  • On new PCR samples, try both iGEM and biobrick RD + Ligations
  • Vicki: iGEM; Marianne: biobrick

Vicki: Protocol from SOP

  • Changes: incubation time 1 hour instead of 2 hours
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)SUPERMIX
Buffer 25x420
BSA0.5x42
ddH2O37x4148
Total42.5170
  • DNA: add 5uL of each C and D and psb1A3 (c,d)
  • Enzymes: EcoRI and Pst I (1uL of each to each tube)

Marianne: Protocol from biobrick (RD)

  • Supermix - same as SOP supermix
  • DNA: add 5uL of each A,B, and psb1A3 (a,b)
  • Enzymes: EcoRI and PstI (1uL each)
  • Changes: 30 min incubation instead of 15 min

Gel verification on RD

  • Protocol: biobrick "digest" protocol
  • Changes: run 10uL instead of 20uL

Gel orientation:

Gel orientation
AaBb100bp ladderCcDd


Ligations

Concentration measurements
TubeConcentration (ng/uL)
A124.9
B123.5
C120.1
D105.6
a82.8
b86.1
c64.7
d64.6

Calculations
 ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)
3 \times \frac{1200}{2400} \times  =  ng
1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} =

  • Where x = concentration of insert

Transformation

  • Protocol: Transformation protocol from SOP
  • Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours
  • Transformants in 37C @ 1815 (3&4)
  • Transformants in 37C @ 1715 (1&2)

Vicki Ma 20:24, 10 August 2010 (EDT)



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