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Latest revision as of 23:16, 18 September 2015


Research Notebook

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


▪ 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


▪ 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.


esa Quorum Sensing    ☀ Yi Han ☀

Re-run gel. 35ul of each sample. No bands.


▪ June 26

esa Quorum Sensing    ☀ Chi Yan & Yun Ting ☀

midiprep of EsaR plasmid


Invasin + Listerolysin    ☀ Yi Han & Yun Ting ☀

Gel electrophoresis of inv colony PCR


esa Quorum Sensing    ☀ Yun Ting & Chi Yan ☀

Midiprep of esaR plasmid. Airdry ON.


▪ June 27

Anaerobic Promoter    ☀ Xin Yi ☀

Colony pcr for placgfp for 18 colonies
▶ POSITIVE COLONIES: 20ul 11 12, 14, 16 17 18


Anaerobic Promoter    ☀ Adrian ☀

gfp expression observed using gfp filter with the SPS microscope