Week 9 (19/7 - 25/7)
▪ July 19
Invasin + Listerolysin
☀ Yi Han ☀
Inoculation of positive colonies in liquid culture. 3mL and shaking incubation.
inoculated colonies C,D for invasin plasmid in 3mL LB+amp at 1.30pm
Transformed remaining 40u ligation reaction for FNR gfp into 10ul BL21
Transformed 100ng pGFPuv plasmid into 10ul BL21
Poured new LB+amp plates
Plated 20, 40, 100ul of transformed bacteria in SOC media on plates
esa Quorum Sensing
☀ Clarice ☀
Colony PCR for RNG and esaR-GFP colonies from master plate
| | RNG (10X) | esaGFP (9X) |
| H2O | 13.8 | 45 |
| Buffer | 50 | 18 |
| MgCl2 | 20 | 18 |
| dNTPs | 20 | 9 |
| Primer 1 | 10 | 9 |
| Primer 2 | 10 | 9 |
| Gotaq | 2 | 1.8 |
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▪ July 20
Invasin + Listerolysin
☀ Yun Ting ☀
Miniprep of positive colonies from 30h liquid culture.
Eluted in 50ul elution buffer and stored at -20.
▶ C= 225.9ng/ul. 260/280=1.86
▶ D= 297.7ng/ul. 1.87
Anaerobic Promoter
☀ Yi Han ☀
1. pGFPuv transformation worked!
2. Single colony for FNR-GFP + 4 more from plating on 19/7
3. Sent FNR-GFP fusion product for sequencing with RP-Biobricks Suffx - result there is no FNR but there is no GFP
4. Colony PCR for FNR gfp (Colonies 1-5, (-) control)
Colony PCR (6X)
| H2O | 19.5ul |
| Buffer | 10ul |
| dNTP | 6ul |
| FP_Biobricks Suffix | 6ul |
| RP_Biobricks SUffix | 6ul |
| MgCl2 | 15ul |
| Taq Polymerase | 1.5ul |
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▪ July 21
Invasin + Listerolysin
☀ Yun Ting ☀
Colony PCR with thermogradient
| H2O | 70ul |
| dNTP | 14ul |
| MgCl2 | 35ul |
| Taq | 3.25ul |
| primers (forward + reverse) | 14*2 |
| Template DNA | 14 |
| C/D-1,2 | Col3. |
| C/D-3,4 | Col12. |
| D-5,6 | Col 9 |
| D-7,8 | Col 10 |
| Negative control (no template) | Col 8 |
▶ Gel ran for 80V, 1h.
D-1,2: VF2, VR.
D-3,4: FP-prefix, RP-suffix.
D-5,6: FP-prefix, FP_M2.
D-7,8: FP-prefix, FP_invF.
C-1,2: VF2, VR.
C-3,4: FP-prefix, RP-suffix.
▶ Results Notes: Too much template DNA (~250ng). Separate the universal primers (to avoid differing extension time).
Anaerobic Promoter
☀ Chi Yan ☀
Ran gel for 20/7 colony pcr for FNR gfp
1kb, 100bp, colonies negative control
Anaerobic Promoter
☀ Yi Han ☀
Redid colony pcr for above as negative control had a positive band - contamination of PCR
negative control still has band - reagents contaminated
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▪ July 22
Anaerobic Promoter
☀ Yi Han ☀
Miniprepped colonies 1-5 for FNR-gfp plasmid
Did EcoRI/PstI digest
| Digest for mastermix (5X) |
| Buffer | 10ul |
| EcoRI/PstI | 2ul |
| H2O | 60ul |
| Total | 20ul per tube |
| (400ng plasmid in each tube) |
Ran gel for RE samples, none were positive clones
Anaerobic Promoter
☀ Chi Yan ☀
PCR to test ‘FNR-GFP plasmid’. Colonies 1-5 from 20/7 + negative control
3 sets of primers
1. FP_BB Prefix, RP BB Suffix
2. GFP-F, GFP-R
3. FP_FNRPromoter_BBP, ORP_FNR_Promoter GFP
| For each reaction (6X) |
| H2O | 73.5ul |
| dNTPs | 6ul |
| Forward/Reverse Primer | 6ul/6ul |
| MgCl2 | 15ul |
| Taq polymerase | 1.5ul |
| Plasmid | 1ul |
| Buffer | 30ul |
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▪ July 23
Invasin + Listerolysin
☀ Yan Ting ☀
RE digest of inv/hyl plasmid with EcoRI and PstI for 2 hours at 37oC.
▶ Lane 1-4: 1kb ladder, 100bp ladder, RE of colony C, RE of colony D.
▶ Size is correct: 4kb main band (inv+hly part) and 2kb (plasmid backbone). These plasmid DNA should have the part.
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▪ July 24
Invasin + Listerolysin
☀ Yun Ting & Yan Ting ☀
Colony PCR, used with temperature gradient to vary annealing temperature.
| H2O | 127.5 |
| 5x buffer | 50 |
| dNTP | 10 |
| MgCl2 | 25 |
| Taq | 2.5 |
| FP + RP | 10*2 |
| Template DNA | 75ng per rxn |
100V for 45min (can run for longer).
Lane 1: 1kb ladder; lane 2: 100bp ladder;
lane 3-7: FP prefix + RP suffix (5 repeats with increasing annealing temperatures) has 2 bands ~800 bp & 100-200bp (probably non specific bands, will lower # of cycles in future, use higher annealing temp, lower annealing time).
Expected size of inv+hly part is 4.1kb.
lane 8-9: VF + M2 has a thick band 800-900bp.
Expected size is 600+130bp (VF adds ~130bp compared to FP_prefix) .: doesn’t seem to be correct….
lane 10-11: VF + inv F has no bands. Expected is at least 200bp (bcos universal primers).
lane 12: VR + inv F seems to have a small band <100bp. Non specific amplification?
Expected is 1.5+0.1 kb (from VR).
Colony PCR for inv/hly plasmid using Biobricks Prefix and Suffix primers, and Universal primers VF, M2 (Using gradient PCR and different annealing temperature) for 5 samples
Ran gel in same order with 1kb, 100bp followed
Anaerobic Promoter
☀ Chi Yan ☀
Ligation of Cut FNRGFP (17/7) with cut GFP vector
| 1X reaction |
| Buffer | 2ul |
| 200ng vector | 1.1ul |
| 3:1 | 256.5ng |
| Water | 3.4ul |
| T4 DNA ligase | 1ul |
| Total | 20ul |
▶ Incubation at room temperature for 2h
▶ Transformation into 10ul BL21 competent cells
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