Team:Heidelberg/k4l1um

From 2009.igem.org

(Difference between revisions)
(→6-18-2009)
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* DNA synthesis for Fos (proximal) and Fos (core)
* DNA synthesis for Fos (proximal) and Fos (core)
* Transformation of DH5a with pcDNA/FRT ΔPstI
* Transformation of DH5a with pcDNA/FRT ΔPstI
 +
Gave many 1000 colonies. Probably a contamination (?), whci hwould explain why there was no mutagenesis (see below)
* HeLa cells, MCS7 and U20S were splitted 1:3. Therefore DEMED medium (containing additionally 10% FCS, Pen-Strep, glutamate and non-essential aminoacids) was removed. Cells were washed afterwards with HBSS and trypsinised with 1 ml of trypsin and incubated for 5 min. at 37°C 5% CO2. The used flasks were filled up to a final volume of 7 ml with DMEM. 2 ml of the suspension was transfered to a new flask, 5 ml DMEM was added and incubated for another 3-4 days at 37°C and 5% CO2.
* HeLa cells, MCS7 and U20S were splitted 1:3. Therefore DEMED medium (containing additionally 10% FCS, Pen-Strep, glutamate and non-essential aminoacids) was removed. Cells were washed afterwards with HBSS and trypsinised with 1 ml of trypsin and incubated for 5 min. at 37°C 5% CO2. The used flasks were filled up to a final volume of 7 ml with DMEM. 2 ml of the suspension was transfered to a new flask, 5 ml DMEM was added and incubated for another 3-4 days at 37°C and 5% CO2.
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 +
== 6-22-2009 ==
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 +
Lab: LV, SH
 +
* Split cells (Split again Thursday!)
 +
* Prepare 6well-plate with U20S cells for Zeomycine assay
 +
* Miniprep  pcDNA/FRT ΔPstI
 +
* Digest with PstI => No mutation
 +
* Use Stratagene QuickChange XL kit for mutagenesis PCR (mcherry, pcDNAand Supercompetent Gold TOP10 cells for transformation => reasonable amount of colonies
 +
 +
== 6-23-2009 ==
 +
 +
Lab: LV, SH
 +
* Added Zeomycine to 6-well platees
 +
* Miniprep mutagenisis PCR
 +
* Digest with PstI => Mutagenesis successful
 +
* PCR to remove EcoRI from pcDNA5/FRT using Phusion polymerase
 +
 +
== 6-24-2009 ==
 +
 +
Lab: LV, SH
 +
* DpnI digest
 +
* Transformation of Supercompetent Gold TOP10 cells with  pcDNA5 ΔEcoRI ΔPstI
 +
 +
== 6-25-2009 ==
 +
'''Split cells!'''
 +
 +
== 6-26-2009 ==
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'''Change 6well plate medium!'''

Revision as of 08:38, 24 June 2009

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 
Gave many 1000 colonies. Probably a contamination (?), whci hwould explain why there was no mutagenesis (see below)
  • HeLa cells, MCS7 and U20S were splitted 1:3. Therefore DEMED medium (containing additionally 10% FCS, Pen-Strep, glutamate and non-essential aminoacids) was removed. Cells were washed afterwards with HBSS and trypsinised with 1 ml of trypsin and incubated for 5 min. at 37°C 5% CO2. The used flasks were filled up to a final volume of 7 ml with DMEM. 2 ml of the suspension was transfered to a new flask, 5 ml DMEM was added and incubated for another 3-4 days at 37°C and 5% CO2.

6-22-2009

Lab: LV, SH

  • Split cells (Split again Thursday!)
  • Prepare 6well-plate with U20S cells for Zeomycine assay
  • Miniprep pcDNA/FRT ΔPstI
  • Digest with PstI => No mutation
  • Use Stratagene QuickChange XL kit for mutagenesis PCR (mcherry, pcDNAand Supercompetent Gold TOP10 cells for transformation => reasonable amount of colonies

6-23-2009

Lab: LV, SH

  • Added Zeomycine to 6-well platees
  • Miniprep mutagenisis PCR
  • Digest with PstI => Mutagenesis successful
  • PCR to remove EcoRI from pcDNA5/FRT using Phusion polymerase

6-24-2009

Lab: LV, SH

  • DpnI digest
  • Transformation of Supercompetent Gold TOP10 cells with pcDNA5 ΔEcoRI ΔPstI

6-25-2009

Split cells!

6-26-2009

Change 6well plate medium!

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