Team:Heidelberg/k4l1um

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Contents

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


LV:

  • Extraction of CMV promoter from 2008 distribution
  • Transformation of DH5a cell with CMV promoter
  • portzughtr

6-16-2009

Lab: LV, SH, CZ

  • DNA synthesis (JeT, cFos, Min)
  • Purification of cDNA via 2% / 3% agarosegel

=> Only bands at ca. 50 Bp => no successful amplification

6-17-2009

Lab: LV, SH

  • No transformations could be observed
  • Replace Phsuion stocks to Phusion Master Mix from Nathan
  • Repeat DNA synthesis 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
  • Annealing successful for JeT and Min
Annealing might not have been successful with Fos beacuse it contains a very repetetive proximal promoter.
  • gel purfifcation of JeT and Min
  • Repeat PCR of pcDNA5/FRT and mcherry with lower annealing temperature (58°C) and fresh Polymerase: Follow PCR protcol from Strategene site directed mutagenesis kit.
mcherry PCR might not have been successful because we used a template we didn't make ourselves - does it contain TE?
  • PCR worked for pcDNA5FRT but not for mcherry
  • DpnI digest of pcDNA5FRT 1,3,4,6

6-18-2009

Lab: LV, SH

  • DNA synthesis for Fos (proximal) and Fos (core)
  • Transformation of DH5a with pcDNA/FRT ΔPstI
  • HeLa cells, MCS7 and U20S were splitted 1:3. Therefore DEMED medium (containing additionally 10% FCS, PenStrap, glutamate and non-essential aminoacids) was removed. Cells were washed afterwards with HGSS and trypsinised with 1 ml of trypsin and incubated for 5 min. at 37°C 5% CO2. The used flasks were filled up to an endvolume of 7 ml with DMEM. 2 ml of the suspension was transfered to a new flask and incubated for another 3-4 days at 37°C and 5% CO2.