IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/06

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cPCR of ASEM 16 to ASEM 21(5 assemblies)

  • performed using standard UBC iGEM cPCR protocol (cultures for ASEM 16 - 21 were in triplicate)
  • primers
  • Forward (FW): VF2
  • Reverse (RE): VR2
  • reagents for each reaction (μL)
  • 10x reaction buffer: 2.5
  • 10μM Forward primer: 1.25
  • 10μM Reverse primer: 1.25
  • 10mM dNTP: 0.5
  • sdH20: 18.8
  • liquid culture: 1.0 (transferred using autoclaved wooden stick)
  • PCR steps [temperature | time]
  • Initial denaturation: 94°C | 120s
  • Denaturation: 94°C | 30s
  • Annealing: 56°C | 30s
  • Extension: 72°C | 72s
  • Final extension: 72°C | 216s
  • Step 2-4 repeated 30 cycles


Gel electrophoresis of ASEM 16 to ASEM 21 cPCR samples

triplicates were labeled a,b,c

  • ASEM 16:
  • Expected band size: 405
  • Conclusion: actual band sizes too large
  • ASEM 17:
  • Expected band size: 1179
  • Conclusion: actual band sizes too large; 17c was correct -> miniprep
  • * ASEM 18
  • Expected band size: 1100
  • Conclusion: 18a and c sizes correct -> miniprep the brighter band
  • ASEM 19
  • Expected band size: 1137
  • Conclusion: correct band size for 19a and b-> miniprep
  • ASEM 20
  • Expected band size: 1178
  • Conclusion: correct band size for 20b-> miniprep
  • ASEM 21
  • Expected band size: 371
  • Conclusion: correct band size for 21b
  • water control: no band -> good, since it's the negative control

ASEM1(C), 12(C), control-lock, key 1 sent for sequencing

Miniprep of ASEM 17, 18, 19, 20, and 21

Using RAf's Alkaline Lysis protocol

  • Sample ID | A260 | A280| 250/280
  • ASEM 17 | 44.895 | 22.389 | 2.01
  • ASEM 18 | 48.223 | 23.256 | 2.07
  • ASEM 19 | 40.835 | 20.418 | 2.00
  • ASEM 20 | 39.314 | 19.067 | 2.06
  • ASEM 21 | 46.664 | 23.009 | 2.03

Diluted all miniprep to 100μg/μL

Addition of LVA tag to Cre through PCR

Expected length: 1.2 kb

  • primer name

Forward (FW): cre-lva fw Reverse (RE): cre-LVA re

  • reagents for each reaction
  • 10x reaction buffer: 2.5
  • 10μM Forward primer: 1.25
  • 10μM Reverse primer: 1.25
  • 10mM dNTP: 0.5
  • sdH20: 0.2
  • DNA: 0.5
  • PCR steps [temperature | time ]
  • Initial denaturation: 94°C | 30s
  • Denaturation: 94°C | 30s
  • Annealing: 68°C | 30s
  • Extension: 72°C | 1min12s
  • Final extension: 72°C | 3min36s
  • Step 2-4 repeated 30 cycles

Gel electrophoresis of PCRed Cre-LVA

successful

Purification of PCR amplified product

using Invitrogen ChargeSwitch kit according to manufacturer's instructions

DNA stored at -20°C

Annealing [lox] (reversed)

1. add 40μL of RNase-free Duplex Buffer to each of the stands to make 100μM stocks 2. add 90μL of RNase-free Duplex Buffer to 10μL of 100μM stocks to make 10μM working solutions 3. add 50μL of each working solution together in a PCR tube; put into PCR machine (heat up to 95°C, ramp down to 24°C at 3% ramp rate)

Assembly (2, 9, 10, 11, 13, 27, 28, and lox into Amp construction plasmid pSB1A3)

1. Digestion, 1h at 37°C using standard UBC iGEM digestion protocol

  • Prefix samples (cut with EcoRI and SpeI)
  • BBa_J23100 °C
  • Lock 1 (Amp)
  • BBa_I13453
  • Suffix samples (cut with XbaI and PstI)
  • BBa_J31001
  • BBa_E0034
  • BBa_E1010
  • BBa_E0022
  • BBa_K145015
  • Backbone cut (cut with PstI and EcoRI)
  • pSB1C3


2. 3A assembly ligation into pSB1A3, 16°C

  • BBa_K145015 and BBa_J31001 were inserted into the backbone
  • BBa_K145015 ligation into plasmid
  • Suffix Part: 8.79ul
  • 10x Buffer: 2ul
  • Ligase: 1ul
  • Vector: 4ul
  • Water: 2.99ul
  • BBa_J31001 ligation into plasmid
  • Suffix Part: 7.09ul
  • 10x Buffer: 2ul
  • Ligase: 1ul
  • Vector: 4ul
  • Water: 4.69ul

To be transformed by Amelia

3. Putting Cre-LVA and lox into Amp plasmid (pSB1A3) 1. digest Cre-LVA and pSB1A3 with EcoR1 and Pst1 2. ligation

  • Ligation reaction mixture:

A. For Lox

  • vector: 7.2ul
  • insert: 1.0ul
  • water:3.3ul
  • 10x buffer: 2ul
  • ligase: 1ul

B. For Cre-LVA

  • vector: 3.0ul
  • insert: 30.0ul
  • water:11.0ul
  • 10x buffer: 5ul
  • ligase: 1ul

To be transformed by Amelia

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