Team:Heidelberg/Notebook/Sensing Group/Notebook/5thweek

Monday, 09/01/2008

1.selection for Fusion-1 and Fusion-2 cloning
the DNA of the colonies was extracted with MINIprep, and then screen with test digestion: PstI (NEBuffer 3 + BSA)
- for Fusion-1: if our insert is there: one band of 6101 bp if no inser: two bands: 4388 + 2213 bp
- for Fusion-2: if our insert is there: one band of 6122 bp if no inser: two bands: 4388 + 2213 bp

PCR of LuxQ and two fusion protein constructs

Lyse V. harveyi for 5 min @ 99 °C
PCR with Phusion (template: supernatend of boiled V. harveyi (a), colony (b))
- 5 min @ 98 °C || 20s @ 98 °C | 40s @ 58 °C | 1min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
- 35(34) µl H₂O, 10 µl 5x HF, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Phusion, 0(1) µl DMSO
PCR with Pfu
- 3 min @ 94 °C || 45s @ 94 °C | 45s @ 58 °C | 2min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
- 40(39) µl H₂O, 5 µl 10x Buffer, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Pfu, 0(1) µl DMSO

NO PCR Product

PCR of LuxQ with taq-Polymerase with different DMSO concentrations (0 %, 5 %, 10 %). As template V. harveyi colonies and supernatant of cooked bacteria was used.

No PCR product

PCR of LuxP and LuxQ from Vibrio harveyi genome with Phusion Mastermix (without any annealing temp). Template: 0.2 µl V. harveyi culture incubated over night in LB(not 20% NaCl LB) at 37 °C.
- result: no PCR product
PCR of LuxP and LuxQ from Vibrio harveyi genome with Fementas tag Mastermix
- sample with normal Mg concentration (2 µg) and 6 µg Mg concentration
- 5min @94°C ||30 sec @ 94°C | 30 sec @ 58°C | 2 min @ 72°C || 5 min @ 72°C | 4°C hold (30 cycles)
- template is 0.2 ul water washed Vibrio harveyi cells.
- result: see tomorrow (NO PRODUCT)
incubation of Vibrio harveyi from glycerol stock at 28 over night (medium is 20% NaCl LB)

PCR of LuxP and LuxQ from Vibrio hayveyi genome with Tag Mastermix
- template is 0.3 ul water washed Vibrio harveyi culture from 3rd September sample with DMSO(1 ul 100%DMSO) and without DMSO
- Result: only byproduct

LuxQ PCR product