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

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


 * 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 A. For Lox
 * Ligation reaction mixture:
 * 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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