Team:Heidelberg/Notebook/Material

From 2010.igem.org

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{| class="wikitable sortable" border="0" align="center" style="text-align: left"
{| class="wikitable sortable" border="0" align="center" style="text-align: left"
|-bgcolor=#009be1
|-bgcolor=#009be1
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|+ align="top, left"|'''table 7''': Used Cell Lines.
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|+ align="top, left"| '''table 7:''' Used Cell Lines.
| Cell line||Tissue of origin||Transfection
| Cell line||Tissue of origin||Transfection
|-
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| Hepa 1-6 || liver hepatoma, mouse || medium ||   
| Hepa 1-6 || liver hepatoma, mouse || medium ||   
|-
|-
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|Huh7 || hepatocarcinoma, mouse || medium ||   
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|HuH7 || hepatocarcinoma, mouse || medium ||   
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| primary hepatocytes || liver, mouse|| hard ||  
| primary hepatocytes || liver, mouse|| hard ||  
|}
|}
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===Plasmids===
===Plasmids===
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{| class="wikitable sortable" border="0" align="center" style="text-align: left"
{| class="wikitable sortable" border="0" align="center" style="text-align: left"
|-bgcolor=#009be1
|-bgcolor=#009be1
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|+ align="top, left"|'''table 7''': Used Plasmids.
+
|+ align="top, left"|'''table 7:''' Used Plasmids.
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| ID||Name||Use|| Sequence Information||Reference
+
| Name||Use|| Sequence Information||Reference
|-
|-
|align="right"| pcDNA5 || expression of shRNAs ||CMV_shRNA_polyA  || [http://tools.invitrogen.com/content/sfs/manuals/pcdna5frtto_man.pdf Invitrogen] ||  
|align="right"| pcDNA5 || expression of shRNAs ||CMV_shRNA_polyA  || [http://tools.invitrogen.com/content/sfs/manuals/pcdna5frtto_man.pdf Invitrogen] ||  
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|align="right"| pTRUF3 || virus packaging (dsDNA) || ITR (AAV2) ||  ||  
|align="right"| pTRUF3 || virus packaging (dsDNA) || ITR (AAV2) ||  ||  
 +
|-
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|align="right"| pBSH1 || virus packaging (ssDNA) || ITR (AAV2) ||  ||
|-
|-
|align="right"| pSB1A3 || 3A assembly cloning || Ampicillin Resistance || [http://partsregistry.org/Part:pSB1A3 Parts Registry] ||  
|align="right"| pSB1A3 || 3A assembly cloning || Ampicillin Resistance || [http://partsregistry.org/Part:pSB1A3 Parts Registry] ||  
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|align="right"| pSB1C3 || 3A assembly cloning || Chloramphenicol Resistance || [http://partsregistry.org/Part:pSB1C3 Parts Registry]  ||  
|align="right"| pSB1C3 || 3A assembly cloning || Chloramphenicol Resistance || [http://partsregistry.org/Part:pSB1C3 Parts Registry]  ||  
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|}
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===Other===
 +
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{| class="wikitable sortable" border="0" align="center" style="text-align: left"
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|-bgcolor=#009be1
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|+ align="top, left"|'''table 8:''' Other reagents.
 +
| Name||Suplier|| Cat. No.
|-
|-
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|align="right"| pTRUF3 || ITR (AAV2) || virus packaging (dsDNA) ||  ||
+
| Matrix Reagent Reservoirs || Thermo Scientific || 8094  
|-
|-
-
|align="right"| pTRUF3 || ITR (AAV2) || virus packaging (dsDNA) || ||  
+
| cell culture flasks 75cm2 || TPP || 90076
 +
|-
 +
| Dual-Luciferase® Reporter Assay System || Promega || E1910 
 +
|-
 +
| white 96-well plates, lumitrac200 || Greiner || 655075
 +
|-
 +
| Rapid DNA Ligation Kit 40 || Roche || 11635379001
 +
|-
 +
| Quick ligation kit || NEB || M2200 S
 +
|-
 +
| 15 cm petri dishes || BD falcons||
 +
|-
 +
| Master Mix Phusion, HF || Finnzymes ||
 +
|-
 +
| 6x Loading Dye || Fermentas || 
 +
|-
 +
| Penicilin Streptomycin || Gibco  || 
 +
|-
 +
| T4 DNA ligase || Fermentas  ||
 +
|-
 +
| FuGENE 6 || Roche  ||
 +
|-
 +
| Proteinase K ||  Qiagen || 
 +
|-
 +
| poly-L-lysine || Sigma  || 
 +
|-
 +
| Benzonase (5kU)|| Clontech ||  
 +
|-
|}
|}
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 +
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Latest revision as of 23:14, 27 October 2010

Contents
 

Materials

Kits

table 1: Used kits.
KitsSupplierCatalog Number
HiSpeed® Plasmid Maxi Kit (25)QIAGEN12663
CompactPrep Plasmid Maxi Kit (25)QIAGEN12763
RNeasy® Mini Kit (25)QIAGEN74106
QIAquick® Miniprep Kit (250)QIAGEN27106
QIAquick® Gel Extraction Kit (250)QIAGEN28706
QIAquick® Nucleotid Removal Kit (250)QIAGEN28306
QIAquick® PCR Purification Kit (250)QIAGEN28106
GeneMorph® II EZC Clone Domain Mutagenesis KitStratagene200552-5

Marker

table 2: Used DNA ladders for gel electrophoresis.
NameSupplierRange
GeneRuler™ High Range DNA LadderFermentas10,171-48,502 bp
GeneRuler™ 1 kb Plus DNA LadderFermentas75-20,000 bp

↑

Enzymes

table 3: Used NEB restriction enzymes.
NameNumber/IDNEB buffer1NEB buffer2NEB buffer3NEB buffer4BSA
AflII2750%100%25%100%x
AgeI1100%50%10%75%
ApaI-25%50%0%100%x
AseInot75%100%not
AvrII-100%100%50%100%
BamHI275%100%100%100%x
BamHI-75%100%100%100%x
BclI-50%100%100%75%
BglI-50%75%100%50%
BglII13 10% 75% 100% 10%
BsrGI- 25% 100% 10% 100%x
DpnI 6 100% 100% 75% 100%
DpnI - 100% 100% 75% 100%
EcoRI - 100% 100% 100% 100%
EcoRV - 50% 75% 100% 50% x
FseI - 100% 75% 0% 100%
HindIII- 10 50% 100% 10% 50%
HindIII-50% 100% 10% 50%
KpnI 11 100% 75% 0% 50% x
MfeI-HF™- 75% 50% 10% 100%
NarI 26 100% 75% 75% 100%
NcoI 12 100% 100% 100% 100%
NdeI - 75% 100% 75% 100%
NheI - 100% 100% 10% 100% x
NotI 14 0% 50% 100% 25%
NsiI - 10% 75% 100% 25%
PciI - 50% 75% 100% 50% x
PstI 15 75% 75% 100% 50%
SacI 16 100% 50% 10% 100%
ScaI 18 notnot100%not
SfcI 19 75% 50% 10% 100% x
SfcI-75% 50% 10% 100% x
SpeI - 75% 100% 25% 100% x
SphI - 100% 100% 50% 100%
SspI - 50% 100% 50% 50%
XbaI 23 0% 100% 75% 100% x
XbaI 56 0% 100% 75% 100% x
XhoI24 75% 100% 100% 100% x
XmaI25 25% 50% 0% 100% x

NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue, NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X

Bacteria

table 4: Used E. coli strains.
NamePurposeReference
DH5αAmplification of PlasmidsInvitrogen
SCS110Amplification of unmethylated PlasmidsStratagene
Top10Amplification of PlasmidsInvitrogen

Media and Antibiotics

  • LB (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, ad 1 L ddH2O)
table 5: Used antibiotics.
AntibioticFinal Concentration Solvent
Ampicillin100 µg/mlddH2O
Chloramphenicol25 µg/ml100% Ethanol
Kanamycin50 µg/mlddH2O

↑

Primers & Oligos

Please find our Primers & Oligos on an extra subpage.

Instruments

table 6: Used instruments.
Description Name Firm
Table Centrifuge Microfuge® 18 Centrifuge Beckman CoulterTM
Table Centrifuge MicrofugeR 22® Centrifuge Beckman CoulterTM
Centrifuge Allegra X-12 Beckman CoulterTM
Shaker VORTEXGENIER 2 Scientific Industries, SITM
Heating plate (magnet stirrer) MR Hei-Standard Heidolph
Heatblock QBD4 Grant
Heatblock (shakeing function) Thermomixer comfort Eppendorf
PCR-machine MyCyclerTM thermo cycler BioRad
UV-chamber Transluminator Vilber Lourmat
Scale (fine) PioneerTM PA114C OHAUS
Scale PioneerTM PA4101C OHAUS
Fridge KTP 1750 Premium Liebherr
Freezer GP 1366 Premium Liebherr
-86 Freezer -86°C Ultralow Freezer NUAIRETM
Hood Tischabzug Wesemann® Laboreinrichtung
Draw-off pump Vacuhand control Vacubrand
Incubator HT Multitron Version 2 INFORS
Incubator (cell culture) HERAcell 150 Thermo electron coorperation
Hood (cell culture) HERAsafe Thermo electron coorperation
Plate reader Infinite M200 TECAN
FACS Cytomics FC 500 MPL Beckman CoulterTM
Microscope CKX41 Olympus
Computer Sun Microsystems
UV/Visivle Spectrometer Ultrospec 3300 pro Amersham Biosciences
Photometer NanoDrop® ND-1000 Spectrophotometer peQLab Biotechnologie GmbH
Photometer NanoVueTM General Electric
Ice Machine MF22 SCOTSMAN®
Gel electrophoresis chamber Mupid®-One Advance
Pipetting robot QIAcubeTM QIAGEN
Wide field microscope Nikon Eclipse 90i Nikon
PCR machine MyCycler thermal cycler BIO-RAD
Real-time PCR machine StepOnePlusTM Real-time PCR System Applied BiosystemsTM

Cell lines

table 7: Used Cell Lines.
Cell lineTissue of originTransfection
HeLa P4 epithelial cervical cancer, human easy
HEK 293T embryonic kidney cells, human easy
Flp-In™ T-REx™ 293 embryonic kidney cells, human easy
Hep G2 hepatocellular carcinoma, human medium
Hepa 1-6 liver hepatoma, mouse medium
HuH7 hepatocarcinoma, mouse medium
primary hepatocytes liver, mouse hard

Plasmids

table 7: Used Plasmids.
NameUse Sequence InformationReference
pcDNA5 expression of shRNAs CMV_shRNA_polyA [http://tools.invitrogen.com/content/sfs/manuals/pcdna5frtto_man.pdf Invitrogen]
pcDNA6/TR amplification of Tet Repressor CMV_TetR_polyA [http://tools.invitrogen.com/content/sfs/vectors/pcdna6tr_map.pdf Invitrogen]
PsiCheck2 cloning of tuning construct HSV_hRluc/SV40_hFluc [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1602 Promega]
pBSU6 virus packaging (ssDNA) ITR (AAV2) Addgene
pTRUF3 virus packaging (dsDNA) ITR (AAV2)
pBSH1 virus packaging (ssDNA) ITR (AAV2)
pSB1A3 3A assembly cloning Ampicillin Resistance [http://partsregistry.org/Part:pSB1A3 Parts Registry]
pSB1K3 3A assembly cloning Kanamycin Resistance [http://partsregistry.org/Part:pSB1K3 Parts Registry]
pSB1C3 3A assembly cloning Chloramphenicol Resistance [http://partsregistry.org/Part:pSB1C3 Parts Registry]

Other

table 8: Other reagents.
NameSuplier Cat. No.
Matrix Reagent Reservoirs Thermo Scientific 8094
cell culture flasks 75cm2 TPP 90076
Dual-Luciferase® Reporter Assay System Promega E1910
white 96-well plates, lumitrac200 Greiner 655075
Rapid DNA Ligation Kit 40 Roche 11635379001
Quick ligation kit NEB M2200 S
15 cm petri dishes BD falcons
Master Mix Phusion, HF Finnzymes
6x Loading Dye Fermentas
Penicilin Streptomycin Gibco
T4 DNA ligase Fermentas
FuGENE 6 Roche
Proteinase K Qiagen
poly-L-lysine Sigma
Benzonase (5kU) Clontech


↑

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