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Team:Heidelberg/k4l1um
From 2009.igem.org
(Difference between revisions)
| Line 42: | Line 42: | ||
* Annealing of Oligos (JeT, cFos, Min) | * Annealing of Oligos (JeT, cFos, Min) | ||
* Purification of cDNA via 2% / 3% agarosegel | * Purification of cDNA via 2% / 3% agarosegel | ||
| + | => Only bands at ca. 50 Bp => no successful amplification | ||
| - | * [[Team:Heidelberg/n4tr1um#Site-directed mutagenesis|Site directed mutagenesis]] of pcDNA5/FRT | + | * [[Team:Heidelberg/n4tr1um#Site-directed mutagenesis|Site directed mutagenesis]] of pcDNA5/FRT and mcherry |
* Dpn1 digestion | * Dpn1 digestion | ||
* Transformation of DH5a | * Transformation of DH5a | ||
| + | |||
| + | == 6-17-2009 == | ||
| + | |||
| + | Lab: LV, SH | ||
| + | * No transformations could be observed | ||
| + | * Replace Phsuion stocks to Phusion Master Mix from Nathan | ||
| + | * Repeat Annealing of Oligos with a different protocol: 1:10 - 1:1000 dilution of Oligos, 95° 5', 7* [95° 45'' 58° 45'' 72° 1'], add 1:10 diluted primers, 95° 5' 25* same temperatures | ||
| + | * Repeat PCR of pcDNA5/FRT and mcherry with lower annealing temperature (58°C) and fresh Polymerase | ||
Revision as of 12:29, 18 June 2009
6-15-2009
Lab: LV, SH, CZ
SH:
- Miniprep of GFP template plasmid, pcDNA5/FRT
| Nr | pcDNA5/FRT | GFP |
|---|---|---|
| 1 | 4,7 | 39,7 |
| 2 | 5,9 | 14,9 |
| 3 | 3,7 | 3,8 |
| 4 | 5,0 | 7,8 |
- Maxiprep pcDNA/FRT 188.7ng/µL
LV:
- Extraction of CMV promoter from 2008 distribution
- Transformation of DH5a cell with CMV promoter
- portzughtr
6-16-2009
Lab: LV, SH, CZ
- Annealing of Oligos (JeT, cFos, Min)
- Purification of cDNA via 2% / 3% agarosegel
=> Only bands at ca. 50 Bp => no successful amplification
- Site directed mutagenesis of pcDNA5/FRT and mcherry
- Dpn1 digestion
- Transformation of DH5a
6-17-2009
Lab: LV, SH
- No transformations could be observed
- Replace Phsuion stocks to Phusion Master Mix from Nathan
- Repeat Annealing of Oligos with a different protocol: 1:10 - 1:1000 dilution of Oligos, 95° 5', 7* [95° 45 58° 45 72° 1'], add 1:10 diluted primers, 95° 5' 25* same temperatures
- Repeat PCR of pcDNA5/FRT and mcherry with lower annealing temperature (58°C) and fresh Polymerase
