Team:Saarland/Materials
From 2014.igem.org
Materials
The following section lists the used material in the presented iGEM project:
1. Chemicals
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 |
2. Enzymes
Enzyme | Manufacturer |
---|---|
Lysozyme | Serva |
T4 Ligase | Thermo Scientific |
Taq-Polymerase | New England Biolabs |
Q5 High Fidelity Polymerase (Master Mix) | New England Biolabs |
FastDigest MluI | Thermo Scientific |
FastDigest SacI | Thermo Scientific |
FastDigest SphI | Thermo Scientific |
FastDigest EcoRI | Thermo Scientific |
FastDigest XbaI | Thermo Scientific |
FastDigest SpeI | Thermo Scientific |
FastDigest PstI | Thermo Scientific |
Fast AP | Thermo Scientific |
3. oligonucleotides
Abbreviation | 5´-Sequence-3´ |
---|---|
Generation of has2-gfp fusion gene | |
SOE Has for: | TGTACACGCGTAAATCAAGGAGGTG |
SOE Has rev: | CTCGCCCTTGCTCACCATTACATCTAATACCATATCGTATTGTTGACC |
SOE GFP for: | ATGGTATTAGATGTAATGGTGAGCAAGGGCGAG |
SOE GFP rev: | TGTACGAGCTCTTACTTGTACAGCTCGTCCATGC |
Amplification of genomic DNA of B. megaterium | |
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 |
Quick Change PCR for restriction site mutagenesis | |
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 |
Sequencing | |
pSB1C3 check for: | TGCCACCTGACGTCTAAGAAACC |
pSB1C3 check rev: | ATTACCGCCTTTGAGTGAGCTG |
4. Media and Solutions
Solution | Ingredients | Quantity |
---|---|---|
10 x KPP Buffer | KH₂PO₄ K₂HPO₄ | 2,71 g 12.5 g |
Stacking Gel Buffer | Tris SDS | 6.05 g 0.4 g |
Stacking Gel (4 %) | ddH2O Stacking Gel Buffer | 1193 µl 500 µl |
Separating Gel Buffer | Tris SDS | 18.17 g 0.4 g |
Separating Gel (12%) | ddH2O Separating Gel Buffer | 1350 µl 1000 µl |
5 x SDS Sample Buffer | Tris 2-ME | 1.21 g 3.487 g |
10 x TBE | Trizma-Baze Boric Acid | 162 g 27.5 g |
12 % Proline | Proline to 25 ml ddH2O | 3 g |
2 x AB3 | Antibiotic Medium NO. 3 (Difco) to 200 ml ddH2O | 7 g |
2 x SMM | Maleic Acid NaOH sterile filtered | 1.16 g 0,8 g |
20 % Glucose | Glucose to100 ml ddH2O | 20 g |
3.5 ml CR5-Topagar-Portionen | Solution A Solution B | 1.75 ml 1 ml |
8 x CR5-Salze | K₂SO₄ KH₂PO₄ | 1.25 g 0.25 g |
Loading Dye for Agarose Gels | 25 mM Tris-HCl Adjust pH value to 7.8 with HCl | 4 ml
|
Ampicillin Stock Solution (100 mg/ml) | Ampicillin Ethanol | 1000 mg 5ml |
Chloramphenicol Stock Solution (10 mg/ml) | Chloramphenicol to 10 ml Ethanol | 100mg |
CR5-Topagar | SolutionA:
Succrose | 51.5 g |
LB-Agar | LB-Medium (Sigma Aldrich) Agar | 10 g
6 g
|
LB-Medium | LB-Medium (Sigma Aldrich) to 400 ml ddH2O | 12 g |
PEG-P | PEG-6000 1 x SMM | 20 g 50 ml |
SMMP | 2 x AB3 2 x SMM | 25ml 25 ml |
SOC-Medium | Solution 1: Bacto Tryptone | 20 g |
TB-Medium | Yeast Extrakt Tryptone | 24 g 12 g |
Tetracyclin Stammlösung (10 mg/ml) | Tetracycline to 10 ml Ethanol | 100 mg |
Xylose Solution (250 mg/ml) | Xylose to 10 ml ddH2O | 2.5 g |
5. Equipment
Equipment | Manufacturer |
---|---|
300 ml Erlenmeyer Flask | Schott Duran |
300 ml Baffled Flask | Thermo Scientific |
2000 ml Erlenmeyer Flask | Schott Duran |
Ultra Low Temoerature Freezer | Heraeus |
Freezer | Sigma Aldrich |
Refrigerator | Liebherr |
Autoclave: Systec VX-150 | Systec |
Cover Slips | Roth |
Ice Machine | Ziegra |
Electroporation Cuvettes (gap width 0.1 cm) | Sigma-Aldrich |
Elektroporator: Easyject Prima | EquiBio |
Falcon Tubes: 15 ml | Sarstedt |
Electrophoresis systems: PowerPACTM High-Current Power Supply | Biorad |
Western Blot System: Blotting Device: OwlTM Separation System | Thermo Fisher Scientific |
Fluorescence microscopy: Inverse Fluorescence microscope Axiovert 200M | Zeiss |
Scale (MXX2001) | Denver Instruments |
Micro scale (BP 210 S) | Sartorius |
Gloves: Nitrile | VWR |
Imaging System Chemi DOC XRS+ | Biorad |
Incubator: Innova 4230 | Innova |
Cuvettes: Polystyrol/Polystyrene 10 x 4x 45 mm | Sarstedt |
Magnetic Mixer IKAMAG® RCT | IKA® Labortechnik |
Microliter Pipettes: | Brand |
Microliter Pipette Tips: | Sarstedt |
Microwave: Severin 700 | Severin |
Microscope slides | Roth |
PCR-Cycler: Primus advanced 96 | Peqlab |
pH-Meter: 766 Calimatic | Knick |
Photometer: UV2100 UV/VIS Recording Spectrophotometer | Shimadzu |
Petri Dishes | Sarstedt |
Reusable Glass Media Bottles with Cap | Fisher Scientific |
Reaction Tubes: 1,5 ml | Sarstedt |
Speedvac | UNIVAPO 100H |
Thermomixer 5436 | Eppendorf |
Compartment Dryer | Memmert |
Mini UV-Table (60-ECX-20M) | Peqlab |
Water Bath SUB6 Grant | Biolab |
Vortex Genie 2 | Bender & Hobein |
Centrifuges: Biofuge pico | Heraeus |
6. Kits
Kit | Manufacturer |
---|---|
QIAprep Spin Miniprep Kit | Qiagen |
QIAquick Gel Extraction Kit | Qiagen |
Gibson Assembly TM Cloning Kit | NEB |
DNA Distribution Kit 2014 | iGEM |
Nexttec TM 1-Step DNA Isolation Kit for Bacteria | NexttecTM |
7. Strains
Bakterienstamm | Genotyp | Referenz |
---|---|---|
E. coli TOP10 F` | F'{lacIqTn10(TetR)} mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu) | Invitrogen |
B. megaterium MS941 | ΔnprM | Wittchen et al 1995 |
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