Week 5 (21/6 - 27/6)
▪ June 21
esa Quorum Sensing
☀ Yan Ting ☀
optimizing gel extract protocol
▶ RE GFP from PCR (directly RE)
▶ promega agarose 1% gel
▶ add NaAC in binding buffer
Result:
▶ yield for cut plasmid was 12.8ng/ul
▶ GFP was 8.6ng/ul
Ligation reaction 6 reactions
| Buffer | 12ul |
| Vector | 30ul |
| Insert | 30ul |
▶ 6x ligation result transformed into competent DH5\alpha
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▪ June 23
esa Quorum Sensing
☀ Yun Ting ☀
Performed 4x ligation using RE- digested esa and GFP (Chi Yan, 22/6).
Ligation rxn at room temperature for 30min instead of 10min.
▶ Included vector-only and insert-only controls.
▶ Stored in -20deg.
▶ Will run gel tmr. Insert only control should be same size as gfp product. Same for the other control. Can try to plate vector only to see re-ligation??
Nanodrop:
▶ PlacGFP - 642.8ng/ul. 260/280=3.89
▶ Vector ctrl - 756.6ng/ul. 260/280=4.11
▶ Insert ctrl - 557 ng/ul. 260/280=3.86
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▪ June 25
esa Quorum Sensing
☀ Yun Ting ☀
9am: RE of esa and Gfp pcr product
12pm: Ran 0.8% pre-cast gel at 100V for 45min. 10ul of each sample to 2ul of loading dye.
▶ Gel lanes: 100bp; uncut esa (used esa4 from -20); pLacGFP; vector ctrl; insert ctrl; 1kb.
1pm: Gel electrophoresis.
▶ Cast a thick 1% gel (60ml) with combined wells. [Don't need to cast thick gel next time, takes too long to melt]
▶ Gel run at 100V, 45min. Lanes: 100bp, esa, gfp 1&2, 1kb
▶ Image after cutting is also saved.
6pm: Gel extraction of RE esa & gfp.
▶ Used Promega kit, loaded both gfp bands into one column. Eluted with 30ul water for 5min before centrifugation.
▶ Nanodrop:
Esa- 37.7ng/ul. 260/280= 1.83
Gfp - 25.1ng/ul. 260/280= 1.84
Overnight ligation
6x ligation:
| Buffer | 6ul |
| Vector (37.7ng/ul) | 50ng 7.8ul |
| Insert (25.1ng/ul) | 37.7 ng 6.6ul |
| H20 | 87.6ul |
| Ligase | 6ul |
▶ Ligation reaction run overnight in 16 deg hold in thermocycler - 15h, 8pm - 11am.
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esa Quorum Sensing
☀ Yi Han ☀
Re-run gel. 35ul of each sample. No bands.
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