▪ July 26
esa Quorum Sensing
☀ Yun Ting & Chi Yan ☀
Midiprep of esaR plasmid.
Airdry ON.
esa Quorum Sensing
☀ Adrian ☀
BBPprefix_esaRBS PCR synthesis
| PCR Reaction |
| H2O | 22ul |
| Buffer | 80ul |
| MgCl2 | 32ul |
| dNTP | 32ul |
| FP_Biobricks_Prefix | 16ul |
| ORP_esaRBS_fragsyn | 16ul |
| synpartBBP_esaRBS | 1ul |
| GoTaq polymerase | 2ul |
| Total | 400ul |
| esaRBS_GFP_BBsuffix |
| H2O | 221ul |
| Buffer | 80ul |
| MgCl2 | 32ul |
| dNTP | 32ul |
| RP_BiobricksSuffix | 16ul |
| OFP_esaRBS_GFPver2 | 16ul |
| pSB1A2_BBa_0040 | 1ul |
| GoTaq polymerase | 2ul |
| Total | 400ul |
| PCR for esa |
| H2O | 220ul |
| Buffer | 80ul |
| MgCl2 | 32ul |
| dNTPs | 32ul |
| FP_BB_Prefix | 16ul |
| RP_BB_Suffix | 16ul |
| BBPrefix_esaRBS | 1.2ul |
| esaRBS_GFPBBSuffix | 0.5ul |
| GoTaq | 2ul |
| Total | 400ul |
| PCR for RNG |
| H2O | 220ul |
| Buffer | 80ul |
| MgCl2 | 32ul |
| dNTPs | 32ul |
| FP_BB_Prefix | 16ul |
| RP_BB_Suffix | 16ul |
| BBPrefix_esaRBS | 1.5ul |
| esaRBS_GFPBBSuffix | 0.5ul |
| GoTaq | 2ul |
| Total | 400ul
|
Anaerobic Promoter
☀ Yi Han ☀
No colonies for FNRgfp
| Hotstart fusion PCR (8X reaction) |
| 10X Buffer | 80ul |
| dNTPs | 16ul |
| FP Biobricks Prefix | 8ul |
| RP Biobricks Suffix | 8ul |
| FNR Pcr product | 12ul |
| GFP PCR product | 4ul |
| MgCl2 | 3ul |
esa Quorum Sensing
☀ Adrian, Clarice, Kenneth ☀
REdigest of esaR and RNF after PCR purification and gel extraction
| esaR-gfp RE digest |
| Buffer | 2ul |
| PstI | 1ul |
| EcoRI | 1ul |
| 2ug Template | 5.4ul |
| H2O | 10.6ul |
| Total | 20ul |
| RNG RE digest |
| Buffer | 2ul |
| PstI | 1ul |
| EcoRI | 1ul |
| 2ug Template | 3.9ul |
| H2O | 12.1ul |
| Total | 20ul |
| Ligation of pSB1A2 with esaR and RNG |
| 10X T4 Ligase Buffer | 2ul |
| Vector (pSB1A2 EcoRI/PstI) | 1ul |
| Insert (esaR and RNG) | 5ul |
| H2O | 11ul |
| T4 DNA ligase | 1ul |
| Total | 20ul |
▶ Ligation for 30min at room temperature
|
▪ July 27
Anaerobic Promoter
☀ Yi Han ☀
Ran gel to determine FNR gfp size
(1kb 100bp FNRgfp PCR)
Result: Faint band of correct size
Gel extract
Anaerobic Promoter
☀ Chi Yan ☀
RE of extracted product with EcoRI/PstI for 300ng of DNA
| RE (1X) |
| FNR-gfp | 0.9ul |
| EcoRI | 1ul |
| PstI | 1ul |
| Buffer | 2ul |
| Water | 15.1ul |
| Total | 20ul |
Anaerobic Promoter
☀ Yi Han ☀
Ran gel with cut vector and cut PCR product
▶ 1kb 100bp cut vector (wells 3-5) FNR-gfp (wells 6-8)
▶ However, PCR product was not detectable
| RE digest for FNRgfp EcoRI/PstI |
| 150ng PCR | 5ul |
| Buffer | 2ul |
| EcoRI | 0.5ul |
| PstI | 0.5ul |
| H2O | 12ul |
| Total | 20ul |
▶ Gel extracted
Ligation reaction was performed overnight
| Ligation reaction (1X) |
| 100ng vector | 8.3ul |
| 85ng insert | 16ul |
| T4 ligase | 2ul |
| Buffer | 4ul |
| H2O | 9.7ul |
|
▪ July 28
Maintenance
☀ Yi Han ☀
1. Trialling transformation of 10ul BL21 with 100ng pGFPuv plasmid for workshop. Using 200ul LB broth for the growth in 1hour step
▶ i. 10ul BL21 + 10ul plasmid
▶ ii. Heatshock for 300s
▶ iii. Shake for 1hour at 37 degC 225rpm
▶ iv. Plated 40ul on LB, 40ul on LB+amp plate
2. Transformation of ligation reaction into BL21 with control of cut vector (100ng) into BL21
▶ Plated 40ul on LB+amp plate, 20ul on LB plate
|
▪ July 29
Maintenance
☀ Yi Han ☀
Colonies on pGFPuv plate with amp -> LB works for reviving bacteria
small colonies with FNRGFP plasmid
|
▪ July 30
Anaerobic Promoter
☀ Yi Han ☀
12 colonies grew for FNRGFP plate
No colonies on control with cut vector
Colony PCR to screen 12 colonies and negative control using universal primers
| PCR Mastermix (13X) |
| H2O | 42.25ul |
| PCR Buffer | 65ul |
| VF2 Primer | 13ul |
| RP VR Primer | 13ul |
| MgCl2 | 32.5ul |
| GoTaq | 3.25ul |
|
▪ July 31
Anaerobic Promoter
☀ Chi Yan & Yi Han ☀
When gel was run, primer dimers and 700-800bp band were present in the negative control with no template added
Fresh buffer, MgCL2, dNTP and fresh diluted primers were used to repeat colony PCR.
Diluting dATP, dCTP, dGTP, dTTP to 2.5mM
Repeated colony pcr with Prefix-suffix primers using TC pipettors
|
▪ Aug 1
esa Quorum Sensing
☀ Chi Yan & Yi Han ☀
esaR PCR check
| PCR reaction |
| H2O | 222ul |
| Buffer | 80ul |
| MgCl2 | 32ul |
| dNTPs | 32ul |
| VF2 | 16ul |
| VR | 16ul |
| GoTaq | 2ul |
▶ Cycle conditions
95ºC (3min) -> 95ºC (30s) -> 60ºC (30s) -> 72ºC (1.5min) -> 72ºC (5min)
for 25 cycles
▶ Result: FP_Biobricks_Suffix and RP_Biobrick_Suffix primers are contaminated -> rediute from stock
|
