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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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| - |
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| - | == 6-16-2009 ==
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| - |
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| - | Lab: LV, SH, CZ
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| - |
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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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| - |
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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|none|frame|Lanes 1-6 Fos (403 Bp); Lanes 7-12 JeT (227 Bp): Lanes 13-18: Min (207 Bp) Lanes 1 : 1µL of 1 :10 diluted oligo 2 : 1 :100 3 : 1 :1000 ;4-6 same, but + DMSO Lanes 7,8,13,14: 1:10 Lanes 9,10,15,16: 1:100 Lanes 11,12,17,18: 1:1000 Even lanes: No DMSO Uneven lanes: DMSO]]
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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
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| - | 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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| - |
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| - | == 6-18-2009 ==
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| - |
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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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| - |
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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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| - |
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| - | == 6-23-2009 ==
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| - |
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| - | Lab: LV, SH
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| - | [[Image:Bild2.jpg|none|frame|2: pcDNA S-1; 3: pcDNA S-4; 4: pcDNA S-2; 5: pcDNA S-3 6:pcDNA S-5 7: pcDNA S-6 8: pcDNA S-7 9: pcDNA S-8; 10: mCherry D-1 11: mcherry S-2; 12: mcherry S-3; 13: pcDNA D-1 14: pcDNA D-2 '''15: pcDNA control 16: mcherry control''']]
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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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| - |
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| - | == 6-26-2009 ==
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| - | '''Change 6well plate medium!'''
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