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Team:Heidelberg/Notebook/Sensing Group/Notebook/5thweek
From 2008.igem.org
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== Wednesday, 09/03/2008 == | == Wednesday, 09/03/2008 == | ||
| + | * PCR of LuxP and LuxQ from ''Virio 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 |30 sec @94||30 sec @58||2 min @72|5 min @72| 4 for ever (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''') | ||
== Thursday, 09/04/2008 == | == Thursday, 09/04/2008 == | ||
Revision as of 15:51, 26 October 2008
Contents |
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 (Buffer3+BSA at 37)
- 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
Tuesday, 09/02/2008
- 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
Wednesday, 09/03/2008
- PCR of LuxP and LuxQ from Virio 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 |30 sec @94||30 sec @58||2 min @72|5 min @72| 4 for ever (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)
