IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/13

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dspB Track

PCR off gDNA for more PCR products

PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x7.537.5
10mM dNTP1x7.57.5
sdH2O6.65x7.549.875
Phusion polymerase0.1x7.5.75
MgCl22x7.515
DMSO - 5%1.25x7.59.375
Total21157.5
PCR Tubes
TubeContent
A1,A2,A3dspB his
B1,B2,B3dspB no-his
W (H2O control)
  • To A1,A2,A3: add 2uL of up-his & dw primers each
  • To B1,B2,B3: add 2uL of up & dw primers each

PCR Cycles:

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

Start: 0907
End: 1010

Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 1% agarose gel
  • Machine conditions: 0.5x TBE buffer, 100V, 45min

Gel orientation:

Gel orientation
A1A2A3100bp ladderB1B2B3W (control)

Results:

O/N cultures

  • 2 tubes (3T1, 4T3) taken out at 0950

Miniprep

  • Miniprepped 3T1, 4T3
  • Protocol: in SOP binder (Alkaline lysis)

Restriction Digest on miniprepped DNA

  • See RD protocol (BioBricks)

Gel Verification on mini-prepped RD

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 1% agarose gel
  • Machine conditions: 0.5x TBE buffer,

Gel orientation:

Gel orientation
3T1100bp ladder4T3chlor

Results:

Plan: RD + ligation + transformation

Restriction Digest

Protocol: Biobrick Protocol

Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)Vicki's SUPERMIX
Buffer 25x1365
BSA0.5x136.5
ddH2O37x13481
Total42.5552.5
  • DNA: add 5uL of backbone + 2uL of insert each
  • Enzymes: EcoRI and SpeI (1uL each to each tube)
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)Marianne's SUPERMIX
Buffer 25x33165
BSA0.5x3316.5
ddH2O37x331221
Total42.51402.5
  • DNA: add 5uL of backbone + 2uL of insert each
  • Enzymes: EcoRI and SpeI (1uL each to each tube)
  • RD Tubes:
    • AA, TA, CA, AB, TB, CB, 1-16 all contain insert dspB His
    • AC, TC, CC< AD, TD, CD, 17-32 all contain insert dspB no-His

Ligation

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

Vicki: ligation - 6:1 ratio using

  1. amp backbone
  2. amp+tet backbone
  3. chlor backbone
Calculations
TubeAmount of vector(uL)Amount of insert (uL)
A32.559
B32.808
C32.820
D33.042

Marianne: Ligation - chlor backbone using ratios:

  • 2:1,3:1,4:1,6:1,7:1,8:1,9:1,10:1
Calculations for ratio 2:1
TubeAmount of vector(uL)Amount of insert (uL)
A230.852
B230.936
C230.941
D231.014
Calculations for ratio 3:1
TubeAmount of vector(uL)Amount of insert (uL)
A331.2801
B331.4046
C331.4106
D331.5216
Calculations for ratio 4:1
TubeAmount of vector(uL)Amount of insert (uL)
A431.707
B431.872
C431.881
D432.028
Calculations for ratio 6:1
TubeAmount of vector(uL)Amount of insert (uL)
A632.559
B632.808
C632.820
D633.042
Calculations for ratio 7:1
TubeAmount of vector(uL)Amount of insert (uL)
A732.988
B733.279
C733.291
D733.552
Calculations for ratio 8:1
TubeAmount of vector(uL)Amount of insert (uL)
A833.414
B833.747
C833.762
D834.059
Calculations for ratio 9:1
TubeAmount of vector(uL)Amount of insert (uL)
A933.840
B934.215
C934.233
D934.566
Calculations for ratio 10:1
TubeAmount of vector(uL)Amount of insert (uL)
A1034.266
B1034.583
C1034.701
D1035.073

Transformation

  • Protocol: Transformation protocol from SOP
  • Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours

Biofilm Track

Eric F. + Melody

8325-4 Biofilm Growth

Streaked a TSA plate with 8325-4 TSB O/N culture and put in incubator at 37°C

RN4220 Biofilm Growth

Completed Day 3 of the protocol for both plates

  • Test #1 - control E5 showed growth
  • Test #2 - controls B2, B11, C5, E7 and G2 showed growth
  • We decided to continue the experiment as a wet run (due to the contamination the results are meaningless)
    • Next time we will perform the inoculation step in the bio safety cabinet
  • The 60°C air dry step was completed using a heat block
  • For the overnight step, we placed the plates in the bio safety cabinet




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