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Team:Heidelberg/Notebook/Sensing Group/Notebook/5thweek
From 2008.igem.org
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== Monday, 09/01/2008 == | == 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 H<sub>2</sub>O, 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 H<sub>2</sub>O, 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 == | == Tuesday, 09/02/2008 == | ||
Revision as of 15:46, 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
