Team:Heidelberg/k4l1um

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== 6-15-2009 ==
 
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Lab: LV, SH, CZ
 
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'''SH''':
 
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* [[Team:Heidelberg/n4tr1um#Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)|Miniprep]] of GFP template plasmid, pcDNA5/FRT
 
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{| class="wikitable" border="1"
 
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|-
 
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!  Nr
 
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!  pcDNA5/FRT
 
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!  GFP
 
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|-
 
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|  1
 
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|  4,7
 
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|  39,7
 
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|-
 
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|  2
 
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|  5,9
 
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|  14,9
 
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|-
 
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|  3
 
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|  3,7
 
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|  3,8
 
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|-
 
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|  4
 
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|  5,0
 
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|  7,8
 
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|-
 
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|}
 
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* [[Team:Heidelberg/n4tr1um#Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)|Maxiprep]] pcDNA/FRT 188.7ng/µL
 
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'''LV''':
 
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* Extraction of CMV promoter from 2008 distribution
 
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* [[Team:Heidelberg/n4tr1um# Transformation of Bacteria|Transformation]] of DH5a cell with CMV promoter
 
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* portzughtr
 
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== 6-16-2009 ==
 
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Lab: LV, SH, CZ
 
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* DNA synthesis (JeT, cFos, Min)
 
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* Purification of cDNA via 2% / 3% agarosegel
 
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=> Only bands at ca. 50 Bp => no successful amplification
 
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* [[Team:Heidelberg/n4tr1um#Site-directed mutagenesis|Site directed mutagenesis]] of pcDNA5/FRT and mcherry
 
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* Dpn1 digestion
 
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* Transformation of DH5a
 
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== 6-17-2009 ==
 
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Lab: LV, SH
 
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* No transformations could be observed
 
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* Replace Phsuion stocks to Phusion Master Mix from Nathan
 
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* 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
 
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* Annealing successful for JeT and Min
 
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Annealing might not have been successful with Fos beacuse it contains a very repetetive proximal promoter.
 
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[[Image:Bild1.jpg]]
 
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* gel purfifcation of JeT and Min
 
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* 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.
 
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mcherry PCR might not have been successful because we used a template we didn't make ourselves - does it contain TE?
 
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* PCR worked for pcDNA5FRT but not for mcherry
 
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* DpnI digest of pcDNA5FRT 1,3,4,6
 
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== 6-18-2009 ==
 
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Lab: LV, SH
 
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* DNA synthesis for Fos (proximal) and Fos (core)
 
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* Transformation of DH5a with pcDNA/FRT ΔPstI
 
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Gave many 1000 colonies. Probably a contamination (?), whci hwould explain why there was no mutagenesis (see below)
 
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* 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
 
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* Split cells (Split again Thursday!)
 
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* Prepare 6well-plate with U20S cells for Zeomycine assay
 
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* Miniprep  pcDNA/FRT ΔPstI
 
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* Digest with PstI => No mutation
 
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* Use Stratagene QuickChange XL kit for mutagenesis PCR (mcherry, pcDNAand Supercompetent Gold TOP10 cells for transformation => reasonable amount of colonies
 
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== 6-23-2009 ==
 
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Lab: LV, SH
 
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* Added Zeomycine to 6-well platees
 
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* Miniprep mutagenisis PCR
 
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* Digest with PstI => Mutagenesis successful
 
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* PCR to remove EcoRI from pcDNA5/FRT using Phusion polymerase
 
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== 6-24-2009 ==
 
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Lab: LV, SH
 
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* DpnI digest
 
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* Transformation of Supercompetent Gold TOP10 cells with  pcDNA5 ΔEcoRI ΔPstI
 
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== 6-25-2009 ==
 
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'''Split cells!'''
 
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== 6-26-2009 ==
 
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'''Change 6well plate medium!'''
 

Latest revision as of 12:24, 24 June 2009