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- | <br> | + | <html> |
- | <h1>Materials</h1> | + | <iframe src="//player.vimeo.com/video/109005558" width="500" height="281" frameborder="0" webkitallowfullscreen mozallowfullscreen allowfullscreen></iframe> <p><a href="http://vimeo.com/109005558">Saarland University iGEM 2014 - Campus Tour</a> </p></html> |
- | <br>
| + | |
- | <!-- Inhaltsverzeichnis-->
| + | |
- | | + | |
- | <h2>1. Chemicals </h2>
| + | |
- | <br>
| + | |
- | | + | |
- | {|
| + | |
- | |+
| + | |
- | ! Chemical
| + | |
- | ! Manufacturer
| + | |
- | |-
| + | |
- | | 2 log DNA ladder
| + | |
- | | New England Biolabs
| + | |
- | |-
| + | |
- | | 2-Mercapto Ethanol
| + | |
- | | Merck
| + | |
- | |-
| + | |
- | | Acetic Acid 100%
| + | |
- | | zChL UdS
| + | |
- | |-
| + | |
- | | Agar Kobe I
| + | |
- | | Roth
| + | |
- | |-
| + | |
- | | Agarose
| + | |
- | | Peqlab, Biozym
| + | |
- | |-
| + | |
- | | Ampicillin
| + | |
- | | Roth
| + | |
- | |-
| + | |
- | | APS (Ammoniumpersulfat)
| + | |
- | | zChL UdS
| + | |
- | |-
| + | |
- | | BactoTM Casamino-acids
| + | |
- | | BD
| + | |
- | |-
| + | |
- | | BactoTM Tryptone
| + | |
- | | BD
| + | |
- | |-
| + | |
- | | BactoTM Yeast-Extract
| + | |
- | | BD
| + | |
- | |-
| + | |
- | | Boric Acid
| + | |
- | | Merck
| + | |
- | |-
| + | |
- | | Bovine Serum Albumin (BSA)
| + | |
- | | New England Biolabs
| + | |
- | |-
| + | |
- | | Bromophenol Blue
| + | |
- | | Merck
| + | |
- | |-
| + | |
- | | Calcium Chloride
| + | |
- | | Calbiochem
| + | |
- | |-
| + | |
- | | Chloramphenicol
| + | |
- | | Fagran
| + | |
- | |-
| + | |
- | | D(+)-Glucose
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | ddH2O (MilliQ) (MILLIPORE 0,22 μm Membranfilter)
| + | |
- | | Biocel
| + | |
- | |-
| + | |
- | | DifcoTM Antibiotic Medium No. 3
| + | |
- | | BD
| + | |
- | |-
| + | |
- | | Dimethylsulfoxide (DMSO)
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | dNTP-Mix (10 mM)
| + | |
- | | Peqlab
| + | |
- | |-
| + | |
- | | EDTA
| + | |
- | | Roth
| + | |
- | |-
| + | |
- | | Ethanol, HPLC grade
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | Glutamate
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | Glycerol
| + | |
- | | zChL UdS
| + | |
- | |-
| + | |
- | | Hydrochloric Acid 37 % (HCl)
| + | |
- | | VWR
| + | |
- | |-
| + | |
- | | Immersion Oil
| + | |
- | | Leica
| + | |
- | |-
| + | |
- | | Isopropanol
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | LB-Medium (Miller)
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | Magnesium Chloride Hexahydrate
| + | |
- | | VWR
| + | |
- | |-
| + | |
- | | Magnesium Sulfate
| + | |
- | | zChL UdS
| + | |
- | |-
| + | |
- | | Maleic Acid
| + | |
- | | Roth
| + | |
- | |-
| + | |
- | | Natriumchlorid
| + | |
- | | VWR
| + | |
- | |-
| + | |
- | | Natriumhydroxid
| + | |
- | | Grüssing
| + | |
- | |-
| + | |
- | | PEG-6000
| + | |
- | | Merck
| + | |
- | |-
| + | |
- | | peqGREEN
| + | |
- | | Peqlab
| + | |
- | |-
| + | |
- | | Potassium Chloride
| + | |
- | | Grüssing
| + | |
- | |-
| + | |
- | | Potassium Dihydrogen Orthophosphate
| + | |
- | | Grüssing
| + | |
- | |-
| + | |
- | | Potassium Hydrogen Phosphate
| + | |
- | | Grüssing
| + | |
- | |-
| + | |
- | | Potassium Sulfate
| + | |
- | | zChL UdS
| + | |
- | |-
| + | |
- | | Proline
| + | |
- | | Merck
| + | |
- | |-
| + | |
- | | Protein Standard
| + | |
- | | New England Biolabs
| + | |
- | |-
| + | |
- | | Rotiphorese Gel 30
| + | |
- | | Roth
| + | |
- | |-
| + | |
- | | SDS
| + | |
- | | Applichem/Roth
| + | |
- | |-
| + | |
- | | Succrose
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | Temed
| + | |
- | | Applichem
| + | |
- | |-
| + | |
- | | Tetracycline
| + | |
- | | Serva
| + | |
- | |-
| + | |
- | | Tris-Baze
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | Trizma Base
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | | Xylose
| + | |
- | | Sigma Aldrich
| + | |
- | |-
| + | |
- | |}
| + | |
- | | + | |
- | <br>
| + | |
- | | + | |
- | <h2> 2. Enzymes</h2>
| + | |
- | <br>
| + | |
- | {|
| + | |
- | |+
| + | |
- | !Enzyme
| + | |
- | !Manufacturer
| + | |
- | |-
| + | |
- | | Lysozyme
| + | |
- | | Serva
| + | |
- | |-
| + | |
- | | T4 Ligase
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | Taq-Polymerase
| + | |
- | | New England Biolabs
| + | |
- | |-
| + | |
- | | Q5 High Fidelity Polymerase (Master Mix)
| + | |
- | | New England Biolabs
| + | |
- | |-
| + | |
- | | FastDigest <i>Mlu</i>I
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | FastDigest <i>Sac</i>I
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | FastDigest <i>Sph</i>I
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | FastDigest <i>Eco</i>RI
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | FastDigest <i>Xba</i>I
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | FastDigest <i>Spe</i>I
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | FastDigest <i>Pst</i>I
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | | Fast AP
| + | |
- | | Thermo Scientific
| + | |
- | |-
| + | |
- | |}
| + | |
- | <br>
| + | |
- | | + | |
- | <h2> 3. oligonucleotides </h2>
| + | |
- | <br>
| + | |
- | {|
| + | |
- | |+
| + | |
- | !Abbreviation
| + | |
- | !5´-Sequence-3´
| + | |
- | |-
| + | |
- | | <b>Generation of <i>has2-gfp</i> fusion gene</b>
| + | |
- | |
| + | |
- | |-
| + | |
- | | SOE Has for:
| + | |
- | | TGTACACGCGTAAATCAAGGAGGTG
| + | |
- | |-
| + | |
- | | SOE Has rev:
| + | |
- | | CTCGCCCTTGCTCACCATTACATCTAATACCATATCGTATTGTTGACC
| + | |
- | |-
| + | |
- | | SOE GFP for:
| + | |
- | | ATGGTATTAGATGTAATGGTGAGCAAGGGCGAG
| + | |
- | |-
| + | |
- | | SOE GFP rev:
| + | |
- | | TGTACGAGCTCTTACTTGTACAGCTCGTCCATGC
| + | |
- | |-
| + | |
- | |<b>Amplification of genomic DNA of <i>B. megaterium</i></b>
| + | |
- | |
| + | |
- | |-
| + | |
- | | RBS UDP-GlcDH for
| + | |
- | | TGTACGAGCTCAAATCAAGGAGGTGAATATACAATGACTAATATCACAGTAGCGGGTACTGG
| + | |
- | |-
| + | |
- | | UDP-GlcDH rev:
| + | |
- | | TGTACGCATGCTTAGACATTAGCTTTAATTTTAGCTTTGTATCC
| + | |
- | |-
| + | |
- | | Glk for
| + | |
- | | TGTAC GAATTC ATGGATGACAAATGGTTAG
| + | |
- | |-
| + | |
- | | Glk rev:
| + | |
- | | TGTAC CTGCAG TTA TTATTCAAAATAAGAT
| + | |
- | |-
| + | |
- | | PgmB for
| + | |
- | | TGTAC GAATTC ATGAAAAAAGAAGAGCTAC
| + | |
- | |-
| + | |
- | | PgmB rev:
| + | |
- | |TGTAC ACTAGT TTA TTAATTTTTTGTATAC
| + | |
- | |-
| + | |
- | | GtaB for
| + | |
- | | TGTAC GAATTC ATGACGATAAAAAAGGCAG
| + | |
- | |-
| + | |
- | | GtaB rev:
| + | |
- | | TGTAC CTGCAG TTA TTAGCTGAAGTTGTTTGC
| + | |
- | |-
| + | |
- | | Pgi for
| + | |
- | | TGTAC TCTAGA ATGACACATGTTCGTTTTG
| + | |
- | |-
| + | |
- | | Pgi rev:
| + | |
- | | TGTAC CTGCAG TTA TTATTTCAAACGCTTTTC
| + | |
- | |-
| + | |
- | | GlmS for
| + | |
- | | TGTAC TCTAGA ATGTGCGGAATTGTAGG
| + | |
- | |-
| + | |
- | | GlmS rev:
| + | |
- | | TGTAC CTGCAG TTA TTATTCTACAGTAACTG
| + | |
- | |-
| + | |
- | | GlmM for
| + | |
- | | TGTAC GAATTC ATGGGTAAGTATTTTGG
| + | |
- | |-
| + | |
- | | GlmM rev:
| + | |
- | | TGTAC CTGCAG TTA TTACTCTAAGCCCATTTC
| + | |
- | |-
| + | |
- | | GcaD for
| + | |
- | | TGTAC GAATTC ATGTCAAAAAGATATGC
| + | |
- | |-
| + | |
- | | GcaD rev:
| + | |
- | | TGTAC CTGCAG TTA TTAGGATTTTTTATTAATATC
| + | |
- | |-
| + | |
- | |<b>Quick Change PCR for restriction site mutagenesis</b>
| + | |
- | |
| + | |
- | |-
| + | |
- | | QC Has for:
| + | |
- | | GAATTTGTAGAAGATTGGTACTCTCAAGAATTTATGGGTAACCAG
| + | |
- | |-
| + | |
- | | QC has rev:
| + | |
- | | AAATTCTTGAGAGTACCAATCTTCTACAAATTCGTGTAATAAAGAG
| + | |
- | |-
| + | |
- | | QC UDP-GlcDH for:
| + | |
- | | TAATTATTAAAAACTCTGATGCTGGTAAATTATTTTTCACAACTG
| + | |
- | |-
| + | |
- | | QC UDP-GlcDH rev:
| + | |
- | | ACCAGCATCAGAGTTTTTAATAATTAATTCATCAAGCCCAGG
| + | |
- | |-
| + | |
- | | QC PgmB for:
| + | |
- | | TTGCAGCTGCTGATTGAGTTCTTCAG
| + | |
- | |-
| + | |
- | | QC PgmB rev:
| + | |
- | | CTGCAAGGGATCAGCCGAGTG
| + | |
- | |-
| + | |
- | | QC Pgi for:
| + | |
- | | AAATTCTTCTTTGTCATACTCCGTAGG
| + | |
- | |-
| + | |
- | | QC Pgi rev:
| + | |
- | | GAATTTGCACGCATTCAAAAAAGTGC
| + | |
- | |-
| + | |
- | | QC GlmS for:
| + | |
- | | AAATTCACTTGCTACGTGCACTTCTAC
| + | |
- | |-
| + | |
- | | QC GlmS rev:
| + | |
- | | GAATTTTCTTATAACATGCCGCTTTTATCTG
| + | |
- | |-
| + | |
- | | QC GlmM for:
| + | |
- | | TCGAGATCTGCAAATAAATGTGCTGC
| + | |
- | |-
| + | |
- | | QC GlmM for:
| + | |
- | | TCTCGATGCAGATATCTCTACAATGG
| + | |
- | |-
| + | |
- | |<b>Sequencing</b>
| + | |
- | |
| + | |
- | |-
| + | |
- | | pSB1C3 check for:
| + | |
- | | TGCCACCTGACGTCTAAGAAACC
| + | |
- | |-
| + | |
- | | pSB1C3 check rev:
| + | |
- | | ATTACCGCCTTTGAGTGAGCTG
| + | |
- | |-
| + | |
- | |}
| + | |
- | <br>
| + | |
- | <h2> 4. Media and Solutions </h2>
| + | |
- | <br>
| + | |
- | {|
| + | |
- | |+
| + | |
- | !Solution
| + | |
- | !Ingredients
| + | |
- | !Quantity
| + | |
- | |-
| + | |
- | |<b>10 x KPP Buffer</b>
| + | |
- | |KH₂PO₄<br>
| + | |
- | K₂HPO₄<br>
| + | |
- | to 100 ml ddH2O<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |2,71 g<br>
| + | |
- | 12.5 g <br> <br><br>
| + | |
- | |-
| + | |
- | |<b>Stacking Gel Buffer</b>
| + | |
- | |Tris<br>
| + | |
- | SDS<br>
| + | |
- | Adjust pH value to 6.7
| + | |
- | to 100 ml ddH2O
| + | |
- | |6.05 g<br>
| + | |
- | 0.4 g<br><br>
| + | |
- | |-
| + | |
- | | + | |
- | |<b>Stacking Gel (4 %)</b>
| + | |
- | |ddH2O<br>
| + | |
- | Stacking Gel Buffer<br>
| + | |
- | Rotiphorese Gel 30<br>
| + | |
- | Temed<br>
| + | |
- | 10% APS<br>
| + | |
- | |1193 µl<br>
| + | |
- | 500 µl <br> | + | |
- | 267 µl<br>
| + | |
- | 5 µl<br>
| + | |
- | 10 µl<br>
| + | |
- | |-
| + | |
- | |<b>Separating Gel Buffer</b>
| + | |
- | |Tris<br>
| + | |
- | SDS<br>
| + | |
- | Adjust pH value to 8.8
| + | |
- | To 100 ml ddH2O
| + | |
- | |18.17 g<br>
| + | |
- | 0.4 g<br> | + | |
- | <br>
| + | |
- | |-
| + | |
- | |<b>Separating Gel (12%)</b>
| + | |
- | |ddH2O<br>
| + | |
- | Separating Gel Buffer<br>
| + | |
- | Rotiphorese Gel 30<br>
| + | |
- | Temed<br>
| + | |
- | 10% APS<br>
| + | |
- | |1350 µl<br>
| + | |
- | 1000 µl<br>
| + | |
- | 1600 µl<br>
| + | |
- | 10 µl<br>
| + | |
- | 20 µl<br>
| + | |
- | |-
| + | |
- | |<b>5 x SDS Sample Buffer</b>
| + | |
- | |Tris <br>
| + | |
- | 2-ME<br>
| + | |
- | SDS <br>
| + | |
- | Glycerol<br>
| + | |
- | Bromophenol Blue<br>
| + | |
- | Adjust pH value to 6.5 with HCl
| + | |
- | to 50 ml ddH2O
| + | |
- | |1.21 g<br>
| + | |
- | 3.487 g<br>
| + | |
- | 5 g<br>
| + | |
- | 10 ml<br>
| + | |
- | 25 mg<br> <br>
| + | |
- | |-
| + | |
- | |<b>10 x TBE</b>
| + | |
- | |Trizma-Baze<br>
| + | |
- | Boric Acid<br>
| + | |
- | EDTA<br>
| + | |
- | to 1000 ml ddH2O
| + | |
- | |162 g<br>
| + | |
- | 27.5 g<br>
| + | |
- | 9.3 g<br><br>
| + | |
- | |-
| + | |
- | |<b>12 % Proline</b>
| + | |
- | |Proline<br>
| + | |
- | to 25 ml ddH2O<br>
| + | |
- | <b>sterile filtered</b>
| + | |
- | |3 g <br> <br><br><br>
| + | |
- | |-
| + | |
- | |<b>2 x AB3</b>
| + | |
- | |Antibiotic Medium NO. 3 (Difco)<br>
| + | |
- | to 200 ml ddH2O<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |7 g <br> <br> <br><br>
| + | |
- | |-
| + | |
- | |<b>2 x SMM</b>
| + | |
- | |Maleic Acid<br>
| + | |
- | NaOH<br>
| + | |
- | MgCl₂ x 6H2O<br>
| + | |
- | Succrose<br><br>
| + | |
- | Solve in 150 ml ddH2O<br>
| + | |
- | Adjust pH value to 6.5 with HCl to 250 ml ddH2O.<br>
| + | |
- | | + | |
- | <b>sterile filtered</b>
| + | |
- | |1.16 g<br>
| + | |
- | 0,8 g<br>
| + | |
- | 2.03 g<br>
| + | |
- | 85.58 g<br>
| + | |
- | <br> <br>
| + | |
- | <br><br> <br>
| + | |
- | |-
| + | |
- | |<b>20 % Glucose</b>
| + | |
- | |Glucose<br>
| + | |
- | to100 ml ddH2O<br>
| + | |
- | <b>steril filtered</b>
| + | |
- | |20 g<br> <br> <br><br>
| + | |
- | |-
| + | |
- | |<b>3.5 ml CR5-Topagar-Portionen</b>
| + | |
- | |Solution A<br>
| + | |
- | Solution B<br>
| + | |
- | 8 x CR5-Salts<br>
| + | |
- | 12 % Proline<br>
| + | |
- | 20 % Glucose
| + | |
- | |1.75 ml<br>
| + | |
- | 1 ml<br>
| + | |
- | 0.346 ml<br>
| + | |
- | 0.175 ml<br>
| + | |
- | 0.175 ml<br>
| + | |
- | |-
| + | |
- | |<b>8 x CR5-Salze </b>
| + | |
- | |K₂SO₄<br>
| + | |
- | KH₂PO₄<br>
| + | |
- | MgCl₂ x 6H₂O<br>
| + | |
- | CaCl₂<br>
| + | |
- | to 625 ml ddH2O<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |1.25 g<br>
| + | |
- | 0.25 g <br>
| + | |
- | 50 g <br>
| + | |
- | 11 g<br><br>
| + | |
- | <br>
| + | |
- | |-
| + | |
- | |<b> Loading Dye for Agarose Gels </b>
| + | |
- | |25 mM Tris-HCl<br>
| + | |
- | Adjust pH value to 7.8 with HCl<br>
| + | |
- | Bromophenol Blue<br>
| + | |
- | Xylene Cyanol<br>
| + | |
- | Glycerol<br>
| + | |
- | EDTA<br>
| + | |
- | |4 ml<br>
| + | |
- | <br>
| + | |
- | 3 mg<br>
| + | |
- | 3 mg<br>
| + | |
- | 6 ml<br><br>
| + | |
- | |-
| + | |
- | |<b>Ampicillin Stock Solution (100 mg/ml)</b>
| + | |
- | |Ampicillin<br>
| + | |
- | Ethanol<br>
| + | |
- | ddH2O
| + | |
- | |1000 mg<br>
| + | |
- | 5ml<br>
| + | |
- | 5ml<br>
| + | |
- | |-
| + | |
- | |<b> Chloramphenicol Stock Solution (10 mg/ml)</b>
| + | |
- | |Chloramphenicol<br>
| + | |
- | to 10 ml Ethanol
| + | |
- | |100mg <br> <br>
| + | |
- | |-
| + | |
- | |<b> CR5-Topagar</b>
| + | |
- | |<b>SolutionA:</b>
| + | |
- | Succrose<br>
| + | |
- | MOPS<br>
| + | |
- | NaOH<br>
| + | |
- | to 250 ml ddH2O<br>
| + | |
- | Adjust pH value to 7.3 with NaOH<br>
| + | |
- | <b>Steril filtered</b> <br><br>
| + | |
- | <b>Solution B:</b> <br>
| + | |
- | Agar<br>
| + | |
- | Casamino Acids (Difco)<br>
| + | |
- | Yeast Extract<br>
| + | |
- | to 142.5 ml ddH2O<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |<br><br>
| + | |
- | 51.5 g<br>
| + | |
- | 3.25 g<br>
| + | |
- | 0.3 g<br><br> <br> <br> <br><br>
| + | |
- | 2 g<br>
| + | |
- | 0.1 g<br>
| + | |
- | 5 g <br> <br> <br>
| + | |
- | |-
| + | |
- | |<b> LB-Agar </b>
| + | |
- | |LB-Medium (Sigma Aldrich)<br>
| + | |
- | Agar<br>
| + | |
- | to 400 ml ddH2O<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |10 g
| + | |
- | 6 g
| + | |
- | <br>
| + | |
- | <br><br>
| + | |
- | |-
| + | |
- | |<b> LB-Medium </b>
| + | |
- | |LB-Medium (Sigma Aldrich)<br>
| + | |
- | to 400 ml ddH2O<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |12 g <br> <br> <br>
| + | |
- | |-
| + | |
- | |<b> PEG-P </b>
| + | |
- | |PEG-6000<br>
| + | |
- | 1 x SMM<br>
| + | |
- | <b>autoclaved</b>
| + | |
- | |20 g<br>
| + | |
- | 50 ml<br><br>
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| </div> | | </div> |
| </div> | | </div> |
| + | |
| + | {{Team:Saarland/footer}} |
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