Week 12 (13/9 - 18/9)
▪ Sep 13
Invasin + Listerolysin
Anaerobic Promoter
☀ Chi Yan ☀
Ran gel for placgfp-psB1C3 colonies from Chi Yan 12/9 11pm. 1% 110V 30min
▶ no bands
▶ no positive colonies
Ran gel for PCRs to make Prefix-pNirB-inv-Suffix from Chi Yan 12/9 11pm
▶ E: successful, cut bands.
▶ F, G, H,I no bands
PCR for YT, Template used is gel extract ‘1.5.2’ (26ng/ul)
Primers used are FP2’ and RP2
8 cycles 50-60degrees 2 min for annealing step
30 cycles 55-65 degrees 40s for annealing step
50ul, 3 tubes, put in columns 4, 8 12. Negative control added.
| Buffer | 15 |
| dNTPs | 6 |
| MgCl2 | 6 |
| Primers | 1.5/1.5 |
| Template | 15 |
| H2O | 104.25 |
| Taq | 0.75 |
Invasin + Listerolysin
Anaerobic Promoter
☀ Yi Han ☀
Ran gel for YT PCR
PCR using F2 to produce F2/invendsuffix
Primer pairs are invlloF3new/invendsuffix and invlloF6/invendsuffix
PCR for YT, Template used is gel extract ‘1.5.2’ (26ng/ul)
Primers used are FP2’ and RP2
| invlloF3new/invendsuffix |
| Template (1000ng) | 18 |
| Primer | 2/2 |
| Buffer | 20 |
| sNTP | 8 |
| Taq | 1 |
| H2O | 149 |
| Total | 200 |
| invlloF6/invendsuffix |
| Template 500ng | 9 |
| Primer | 1/1 |
| Buffer | 10 |
| dNTP | 4 |
| Taq | 0.5 |
| H2O | 74.5 |
| Total | 100 |
Attempt to make more of inv fragment from Yun Ting’s gel extract
Primers are FP2’/RP
| Template | 8.2 |
| Primer | 1/1 |
| Buffer | 10 |
| dNTP | 4 |
| Taw | 0.5 |
| H2O | 75.3 |
| Total | 100 |
Invasin + Listerolysin
Anaerobic Promoter
☀ Chi Yan & Yi Han ☀
PCR of invlloF1/lloendBBSuffix R with invasin D plasmid as template
▶ 5 cycles gradient 40-50degrees
▶ 20 cycles 62 degrees
| Buffer | 40 |
| MgCl2 | 16 |
| dNTPs | 16 |
| Primers | 4/4 |
| Template | 9.2 |
| H2O | 308.8 |
| Taq | 2 |
| Total | 400 |
▶ 12 tubes at each temp, 33ul each
For YT: PCR of RP2/FP2’ using gel extract 1.6
| Buffer | 20 |
| dNTP | 8 |
| MgCl2 | 8 |
| Primers | 2/2 |
| Taq | 1 |
| H2O | 135 |
| Total | 200 |
| (3 tubes + 5ul template, 1 tube control) |
Gel extract F1 invlloendsuffix
PCR of invlloend/BBsuffixR with invD
PCR of invlloF2/lloendBBsuffixR with F1/invlloendsuffix
| F1invlloendsuffix |
| BUffer | 80 |
| MgCl2 | 32 |
| dNTPs | 32 |
| Primers | 8/8 |
| Template | 18.4 |
| H2O | 617.6 |
| Taq | 4 |
| Total | 800 |
| Buffer | 20 |
| MgCl2 | 8 |
| dNTPs | 8 |
| Primers | 2/2 |
| Template | 84 |
| H2O | 75 |
| Taq | 1 |
| Total | 200 |
▶ 5 cycles 44-48 degrees
25 cycles 60degrees
Colony PCR new placgfp-pSB1C3 following CY 12/9 11pm
Primers FP_KpnI_GFP/GFP_BBSuffix_new
60 degrees 25 cycles
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▪ Sep 14
Anaerobic Promoter
☀ Yi Han ☀
Ran gel for Chi Yan 13/9 9pm and 11pm
▶ Colony PCR no bands
▶ B1-12 no bands
▶ A1-16 bright band , correct size, extract
Anaerobic Promoter
☀ Chi Yan ☀
PCR of invlloF1/lloendBBsuffixR with invD
| Buffer | 160 |
| MgCl2 | 32 |
| dNTPs | 32 |
| Primers | 32/32 |
| Template | 18.3 |
| H2O | 517.7 |
| Taq | 8 |
| Total | 800 |
PCR of invloF2/lloendBBsuffix R with invlloF1endsuffixR
| Buffer | 80 |
| MgCl2 | 16 |
| dNTPs | 16 |
| Primers | 16/16 |
| Template | 100 |
| H2O | 152 |
| Taq | 4 |
| Total | 400 |
Cycle conditions as of Chi Yan 13/9
Positions of tubes, A-C B-D
PCR done with GoTaq kit
Anaerobic Promoter
☀ Yi Han ☀
Screen placgfp colonies for gfp using microscope
▶ Colonies 1, 4, 8 look promising
Miniprepped placgfp colonies
| RE of colony 1, 4, 8 |
| Template | 1 |
| EcoRI/PstI | 0.1/0.1 |
| Buffer | 1 |
| H2O | 8 |
| Total | 10.2 |
Anaerobic Promoter
☀ Chi Yan ☀
Sent colonies 1, 4, 8 of placgfppSB1C3 for sequencing with FP_BB_Prefix, RP_BB_Suffix, VF2.
Ran gel for YH placgfp pSB1C3 colonies RE 1, 4, 8
Incomplete digestion as only digested for 1.5hours
PCR of placgfp pSB1C3 colonies 1, 4, 8 from plasmids and with placfp pAC with FP_BB_Prefix and RP_BB_Suffix
Annealing temp 55degrees for 30s, extension for 60 seconds, for 25 cycles
| GOTaq Master Mix 1 |
| Buffer | 20 |
| MgCl2 | 10 |
| dNTPs | 4 |
| Primers | 4/4 |
| Taq | 1 |
Anaerobic Promoter
☀ Yi Han ☀
PCR with GoTaq kit to make Fragment 8 from Fragment 1
| Template (500ng) | 40 |
| dNTPs | 4 |
| MgCl2 | 16 |
| Primers | 2/2 |
| GoTaq | 0.5 |
| PCR Buffer | 20 |
| H2O | 19.5 |
| Total | 104.5 |
Continued from previous
Ran gradient at annealing temperature for 55-65, in wells 1, 4, 7, 12
Ran gel, no bands in all
1. Made more F1 invsuffixendloger from invD
2. Use BB PrefixpBirBinvstart/invendBBSuffixend to generate whole fragment
3. Make fragment 8 using F8/invendsuffixlonger
Set PCR machine to 10 cycles at Ta gradient 40-55 degrees
30 cycles Ta 60 degrees
Ramp of 10%
| PCR mix for 1 and 2 |
| InvD Template | 9.2 |
| dNTPs | 20 |
| MgCl2 | 16 |
| Primers | 4/4 |
| GoTaq | 0.5 |
| H2O | 105.8 |
| PCR mix for 3 |
| Template | 45 |
| dNTP | 8 |
| MgCl2 | 8 |
| Primers | 1/1 |
| GoTaq | 1 |
| PCR Buffer | 20 |
| H2O | 16 |
| Total | 100 |
▶ Result: 1, 2 had clear bands, 3 had no bands
Anaerobic Promoter
☀ Chi Yan ☀
PCR of placgfp colonies 1, 4, and 8 plasmids and placgfp pAC colony plasmid using VF2/VR
Annealing temperature is 52degrees for 30seconds, extension of 60s 30 cycles using GoTaq kit
| Buffer | 20 |
| MgCl2 | 16 |
| dNTPs | 4 |
| Primers | 4/4 |
| Taq | 1 |
▶ Colony 1: Template 0.4/12.35 H2O
▶ Colony 4: Template 0.8/11.85 H2O
▶ Colony 8: Template 0.7/12.05 H2O
▶ pAC: Template 2.5/10.25
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▪ Sep 15
esa Quorum Sensing
☀ Adrian ☀
| BBa_B0015 RE D |
| Buffer | 4 |
| H2O | 6 |
| EcoRI-HF | 1 |
| PstI-HF | 1 |
| Template | 10 |
| Total | 20 |
| Colony pcr for two samples |
| H2O | 13.5 |
| Buffer | 5 |
| MgCl2 | 2 |
| dNTP | 2 |
| FP_BBPrefix_Junk | 1 |
| RP_BBPrefix_Junk | 1 |
| Taq | 0.5 |
| Total | 25 |
Rapid DNA Ligation: 200ng insert: 21.4 ng vector
Anaerobic Promoter
☀ Yi Han ☀
PCR with prefix-pNirB ultramer was successful.
| PCR with Junk primers to add sequence |
| dNTPS | 20 |
| MgCl2 | 16 |
| Primers | 4/4 |
| GoTaq | 2 |
| Buffer | 40 |
| Template (1500ng) | 88 |
| H2O | 26 |
| Total | 200 |
PCR 12 tubes, gradient (58-62degrees) for 30cycles
Ran gel first three tubes had PCR product with large bands-gel extract
| RE of above PCR product (Junk-pNirB-inv-suffix-Junk) with EcoRI/PstI |
| DNA | 50 |
| Buffer | 5 |
| EcoRI | 0.5 |
| PstI | 0.5 |
| Total | 55 |
esa Quorum Sensing
☀ Kenneth ☀
| GFP Thingy |
| Buffer 4 | 2 |
| H2O | 6 |
| EcoRI-HF | 1 |
| PstI-HF | 1 |
| Template | 10 |
| Total | 20 |
Anaerobic Promoter
☀ Chi Yan ☀
Colony PCR (and inoculation in 3mL LB+chl) of psB1C3-placgfp colonies 1, 4, 8, 11-31, placgfppAC, water. 15ul reaction *26 = 650
| Mastermix |
| Buffer | 130 |
| MgCl2 | 104 |
| dNTPs | 26 |
| Primers | 26/26 |
| Taq | 6.5 |
| (H2O 11.75 each) |
| (12.25ul mastermix each tube) |
Run gel for YH 15/9 RE of pSB1C3 (gel purified) and Junk-pref-pNirB-inv-suffix-junk with EcoRI/PstI
Anaerobic Promoter
☀ Yi Han ☀
Extract of the RE digested BB-Prefix-pirB-inv-suffix
Ligation Reaction
| Template (insert) | 60 |
| Ligase | 3 |
| 10X buffer | 3 |
| Cut pSB13 | 1.35 |
PCR to get more Junk-prefix-pNirB-Suffix-junk
| Template (2550ng) | 150 |
| dNTPs | 33 |
| MgCl2 | 25.6 |
| Primer | 6.4/6.4 |
| GoTaq | 3.2 |
| Buffer | 64 |
| Total | 250 |
Anaerobic Promoter
☀ Chi Yan ☀
Run gel for CY 15/9 4pm colony pcr
▶ 1kb, 100bp, 1, 4, 8, 11-23, 100bp, 1kb
▶ 1kb, 100bp, 24-31, PCR Junk-Prefix-placgfp-suffix-Junk, pSC, H2O, 100bp, 1kb
Positive result with colony PCR for colonies 12, 13, 15, 17 (does not glow), 19, 20
▶ Duy used confocal microscopy to check for GFP expression
▶ Sequencing results showed that colonies 12, 13 and 19 has correct sequence.
Anaerobic Promoter
☀ Chi Yan ☀
gel extract Junk-prefix-pNirB-inv-suffix-junk from YH 15/9 7pm
| RE with EcoRI/PstI 2hours at 37 degrees |
| DNA | 30 |
| Buffer | 3 |
| EcoRI | 0.5 |
| PstI | 0.5 |
The ligated plasmid was then transformed into 10ul BL21.
| 7:1 ligation reaction |
| Insert | 10.4 |
| Vector | 1.5 |
| Ligase | 1 |
| Buffer | 2 |
| Water | 5.1 |
| 5:1 ligation reaction |
| Insert | 7.56 |
| Vector | 1.5 |
| Ligase | 1 |
| Buffer | 2 |
| Water | 8.04 |
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