IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/03

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DspB Track

Ligation

  • See protocol: "Ligate" protocol in BioBrick Assembly Manual (in protocol binder)
  • Changes: incubate reaction mix at RT for 20 minutes instead of 15 minutes

Transformation

  • See protocol: "Transformation" protocol (SOP) in Protocol Binder
  • Changes: Added 10uL of ligation mix instead of 1uL; Incubate at 37C for 1 hour instead of 2 hours

Tubes: A, B, C, D, E, F onto Plates: A, B, C, D, E, F, respectively
Put in 37C incubator at 1630

Re-do PCR with Rafael's suggestions

  1. 2uL of MgCl2 in each PCR tube
  2. 2% and 5% DMSO for each tube
  • Protocol: common protocols for PCR
PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1050
10mM dNTP2x1020
sdH2O5.55x1055.5
Phusion polymerase0.2x102
MgCl22x1020
Total14.75147.5
PCR Tubes
LabelContent
AdspB His 1
BdspB His 2
CdspB no His 1
DdspB no His 2
EdspB rxn1 1
FdspB rxn1 2
GdspB rxn2 1
HdspB rxn2 2
IH2O control
  • For 1: 2% DMSO
  • For 2: 5% DMSO
  • To each 1 (A,C,E,G), add 2uL of fw + rev primer each and 0.5uL of DMSO and 0.75uL of H2O, and 5uL of DNA
  • To each 2 (B,D,F,H), add 2uL of fw + rev primer each and 1.25uL of DMSO and 5uL of DNA

PCR Cycles:

  • 98C @ 2min
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1437
End: 1540

Gel Verification

  • See protocol: gel verification protocol in Protocol binder (SOP)
  • Changes: 1.2% agarose gel instead of 0.8%

Gel orientation:

Gel orientation
ABCD100bp ladderEFGHI

Machine conditions: 100V, 60 min, 0.5X TBE Buffer
Results:

Vicki Ma 02:38, 4 August 2010 (EDT)

QS Track

  • Resuspended oligos for PCRing agrCA (with nat RBS), agrC, and agr A in the appropriate amount of sdH2O, according to idt resuspension calculator.
  • First resuspend to 100 μM concentration (stock solution), then dilute 1/10 to 10 μM (working solution)
  • PCR'd according to instructions that came with Phusion polymerase.


PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1155
10mM dNTP2x1122
sdH2O5.55x1155.5
Phusion polymerase0.05x110.55
MgCl22x1012
DMSO1x1011
Total14.75147.5
  • Source of DNA are colonies from a plate containing Staph aureus NCTC 8325.
  • To each of the above 1X solutions, added 1.25 μL of each 10 μM reverse and forward primer.
  • Did this in triplicates for each pair of primers with each PCR tube containing a different Staph aureus colony.
  • Used the same PCR program as the dspB track (used at the same time).
  • Gelled using 1.2% agarose over 45 minutes.
  • No bands showed up. Water control was clear.
  • May have to add more DNA (let colonies grow up more) and add more DMSO. Also, annealing temperature should be checked (use only part that binds to template when using Finnzyme Tm calculator). Raising DMSO concentration to 6% may also help.




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