Team:UC Chile/Cyano/Labbook/week23

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{{UC_Chile}}
{{UC_Chile}}
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Monday:
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*PCR colony (Gibson assembly date 08.03)
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* PCR run
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<table class="wikitable">
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<tr>
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<td>
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</td><td>pBAD
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</td><td>PP Luc (1)
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</td><td>PP Luc (2)
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</td><td>LC Luc (1)
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</td></tr>
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<tr>
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<td>Nuclease-free H20
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</td><td>14
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</td><td>14
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</td><td>14
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</td><td>14
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</td></tr>
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<tr>
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<td>10x FD Buffer
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</td><td>2
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</td><td>2
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</td><td>2
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</td><td>2
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</td></tr>
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<tr>
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<td>Plasmid DNA
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</td><td>2
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</td><td>2
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</td><td>2
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</td><td>2
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</td></tr>
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<tr>
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<td>FD EcoRI
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</td><td>1
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</td><td>1
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</td><td>1
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</td><td>1
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</td></tr>
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<tr>
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<td>FD SpeI
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</td><td>1
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</td><td>0
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</td><td>0
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</td><td>0
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</td></tr>
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<tr>
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<td>FD XbaI
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</td><td>0
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</td><td>1
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</td><td>1
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</td><td>1
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</td></tr></table>
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Name  PCR                    Template          Primer F        Primer R      Length    Additive<br><br>
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1 psigEP -LuxCDEG       PCR (date 08.02)  16.H1         16.H4       4000 8%Dmso<br> 
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2 psigEP -LuxCDEG       PCR (date 08.02) 16.H1         16.H4       4000 5%Dmso<br>  
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3 Pcaa3 -LuxCDEG       PCR (date 08.02) 16.H9         16.H4       4000 5%Dmso<br>  
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4 Pcaa3 -LuxCDEG       PCR (date 08.02) 16.H9         16.H4       4000 8%Dmso<br>  
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5 LuxBrick nP       LuxBrick         17.E1         Suffix.Dig R  600<br>  
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6 LuxBrick (wxAB)       LuxBrick         17.E4         17.E5       2000<br>  
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7 LuxAB (4tap)       LuxBrick         17.D7         17.D10       2000<br>  
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8 ADF-3 Alone       ADF-3         17.E2         17.E3       2000<br>  
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9 BBricking VB (V.fis)  PSB1C3 (RS1) Suffix.Dig F Preffix.Dig R  2000<br>  
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10 BBricking VB (V.fis)  PSB1T3         Suffix.Dig F Preffix.Dig R  2000<br>  
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11 ADF-3_VB       LuxBrick         16.F7         16.F10       3000<br>  
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12 ADF-3+_ VB       LuxBrick         16.F7         16.G2       3000<br>  
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13 ADF-3_VB       LuxBrick         16.F7         16.F10       3000<br>  
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14 ADF-3+_ VB       LuxBrick         16.F7         16.G2       3000<br>
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Notes
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*PCR Tm=56°C.
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*11 and 12 Tm=60°C
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*13 and 14 TouchDown PCR. PCR annealing program 54°C.
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Tuesday:
Tuesday:

Latest revision as of 22:56, 9 August 2012

Cyanolux & SpiderColi - Pontificia Universidad Católica de Chile, iGEM 2012



Monday:

  • PCR colony (Gibson assembly date 08.03)
  • PCR run
pBAD PP Luc (1) PP Luc (2) LC Luc (1)
Nuclease-free H20 14 14 14 14
10x FD Buffer 2 2 2 2
Plasmid DNA 2 2 2 2
FD EcoRI 1 1 1 1
FD SpeI 1 0 0 0
FD XbaI 0 1 1 1


Name PCR Template Primer F Primer R Length Additive

1 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 8%Dmso
2 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 5%Dmso
3 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 5%Dmso
4 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 8%Dmso
5 LuxBrick nP LuxBrick 17.E1 Suffix.Dig R 600
6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000
7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000
8 ADF-3 Alone ADF-3 17.E2 17.E3 2000
9 BBricking VB (V.fis) PSB1C3 (RS1) Suffix.Dig F Preffix.Dig R 2000
10 BBricking VB (V.fis) PSB1T3 Suffix.Dig F Preffix.Dig R 2000
11 ADF-3_VB LuxBrick 16.F7 16.F10 3000
12 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000
13 ADF-3_VB LuxBrick 16.F7 16.F10 3000
14 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000

Notes

  • PCR Tm=56°C.
  • 11 and 12 Tm=60°C
  • 13 and 14 TouchDown PCR. PCR annealing program 54°C.



Tuesday:

  • PCR band recuperation

Name PCR Concentration [ng/µl] 1 and 2 psigEP -LuxCDEG 5.7

  • Digestion of colonies selected in colony PCR constructs C1.1 and C1.2 (Gibson assembly date: 08.01)

C1.1: Band from colony 8 is the right size. Construct succesfully assembled and will be sequenced to confirm. C1.2 : Wrong size (all colonies). Probably due to: the use of mRFP instead of RFP for the Gibson assembly.

  • Gibson results (assembly date: 08.03) and Colony PCR

C1.1: two positive colonies but were the wrong size. C1.2: several positive colonies but were the wrong size. Probably due to: the use of mRFP instead of RFP for the Gibson assembly. Pcaa3 BB: several colonies and with the right size. It will be digested. ADF 3: no colonies.

  • Miniprep and positive colonies digestion (Gibson assembly date: 08.03)

Colony Concentration [ng/µl] 1 52.4 2 45.8 5 38.3 10 62.9

  • Synechocystis PCC6803 transformation results (transformation date: 07.20)

Synechocystis did not grow. Probably due to: they die in the presence of glucose. Synechocystis were reinoculated and e. coli was transformed with psbAB+GFP to further transformation in synechocystis.

  • PCR run (59ºC)


Name PCR Template Primer F Primer R Length Additive 1 Bactomithril 2 Bactomithril 4 Pcaa3 -LuxCDEG sigEP -LuxCDEG 16.H9 16.H4 4000 8%Dmso 5 LuxBrick (No P) LuxBrick 17.E1 Suffix.Dig R 600 5%Dmso 6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000 5%Dmso 7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000 5%Dmso 8 ADF-3 Alone ADF-3 17.E2 17.E3 2000 5%Dmso 9 C1_R_VB C1.1 16.E7 16.E6 4300 10 C1.2_VB C1.1 14.F6 14.F5 5200

Notes

  • 9: amplifies from RS2 to mRFP. Switches Kan to KanR.
  • 10: amplifies from mRFP to RS1. Switches psbAB to psbAB2 (C1.2)
Retrieved from "http://2012.igem.org/Team:UC_Chile/Cyano/Labbook/week23"