Team:Saarland/Materials
From 2014.igem.org
Line 59: | Line 59: | ||
| Merck | | Merck | ||
|- | |- | ||
- | | Acetic Acid 100% | + | | Acetic Acid 100 % |
| zChL UdS | | zChL UdS | ||
|- | |- | ||
Line 101: | Line 101: | ||
| Sigma Aldrich | | Sigma Aldrich | ||
|- | |- | ||
- | | | + | | ddH<sub>2</sub>O (MilliQ) (MILLIPORE 0,22 μm Membranfilter) |
| Biocel | | Biocel | ||
|- | |- | ||
Line 397: | Line 397: | ||
|KH₂PO₄<br> | |KH₂PO₄<br> | ||
K₂HPO₄<br> | K₂HPO₄<br> | ||
- | to 100 ml | + | to 100 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> | <b>autoclaved</b> | ||
|2,71 g<br> | |2,71 g<br> | ||
Line 406: | Line 406: | ||
SDS<br> | SDS<br> | ||
Adjust pH value to 6.7 | Adjust pH value to 6.7 | ||
- | to 100 ml | + | to 100 ml ddH<sub>2</sub>O |
|6.05 g<br> | |6.05 g<br> | ||
0.4 g<br><br> | 0.4 g<br><br> | ||
Line 412: | Line 412: | ||
|<b>Stacking Gel (4 %)</b> | |<b>Stacking Gel (4 %)</b> | ||
- | | | + | |ddH<sub>2</sub>O<br> |
Stacking Gel Buffer<br> | Stacking Gel Buffer<br> | ||
Rotiphorese Gel 30<br> | Rotiphorese Gel 30<br> | ||
Temed<br> | Temed<br> | ||
- | 10% APS<br> | + | 10 % APS<br> |
|1193 µl<br> | |1193 µl<br> | ||
500 µl <br> | 500 µl <br> | ||
Line 427: | Line 427: | ||
SDS<br> | SDS<br> | ||
Adjust pH value to 8.8 | Adjust pH value to 8.8 | ||
- | To 100 ml | + | To 100 ml ddH<sub>2</sub>O |
|18.17 g<br> | |18.17 g<br> | ||
0.4 g<br> | 0.4 g<br> | ||
<br> | <br> | ||
|- | |- | ||
- | |<b>Separating Gel (12%)</b> | + | |<b>Separating Gel (12 %)</b> |
- | | | + | |ddH<sub>2</sub>O<br> |
Separating Gel Buffer<br> | Separating Gel Buffer<br> | ||
Rotiphorese Gel 30<br> | Rotiphorese Gel 30<br> | ||
Temed<br> | Temed<br> | ||
- | 10% APS<br> | + | 10 % APS<br> |
|1350 µl<br> | |1350 µl<br> | ||
1000 µl<br> | 1000 µl<br> | ||
Line 451: | Line 451: | ||
Bromophenol Blue<br> | Bromophenol Blue<br> | ||
Adjust pH value to 6.5 with HCl | Adjust pH value to 6.5 with HCl | ||
- | to 50 ml | + | to 50 ml ddH<sub>2</sub>O |
|1.21 g<br> | |1.21 g<br> | ||
3.487 g<br> | 3.487 g<br> | ||
Line 460: | Line 460: | ||
|<b>Na<sub>3</sub>PO<sub>4</sub> Solution (100 mM)</b> | |<b>Na<sub>3</sub>PO<sub>4</sub> Solution (100 mM)</b> | ||
|Na<sub>3</sub>PO<sub>4</sub> <br> | |Na<sub>3</sub>PO<sub>4</sub> <br> | ||
- | Solve in 80 ml | + | Solve in 80 ml ddH<sub>2</sub>O<br> |
Adjust pH value to 6.5 with H<sub>3</sub>PO<sub>4</sub> | Adjust pH value to 6.5 with H<sub>3</sub>PO<sub>4</sub> | ||
to 100 ml ddH<sub>2</sub>O | to 100 ml ddH<sub>2</sub>O | ||
Line 495: | Line 495: | ||
Boric Acid<br> | Boric Acid<br> | ||
EDTA<br> | EDTA<br> | ||
- | to 1000 ml | + | to 1000 ml ddH<sub>2</sub>O |
|162 g<br> | |162 g<br> | ||
27.5 g<br> | 27.5 g<br> | ||
Line 502: | Line 502: | ||
|<b>12 % Proline</b> | |<b>12 % Proline</b> | ||
|Proline<br> | |Proline<br> | ||
- | to 25 ml | + | to 25 ml ddH<sub>2</sub>O<br> |
<b>sterile filtered</b> | <b>sterile filtered</b> | ||
|3 g <br> <br><br><br> | |3 g <br> <br><br><br> | ||
Line 508: | Line 508: | ||
|<b>2 x AB3</b> | |<b>2 x AB3</b> | ||
|Antibiotic Medium NO. 3 (Difco)<br> | |Antibiotic Medium NO. 3 (Difco)<br> | ||
- | to 200 ml | + | to 200 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> | <b>autoclaved</b> | ||
|7 g <br> <br> <br><br> | |7 g <br> <br> <br><br> | ||
Line 517: | Line 517: | ||
MgCl₂ x 6H2O<br> | MgCl₂ x 6H2O<br> | ||
Succrose<br><br> | Succrose<br><br> | ||
- | Solve in 150 ml | + | Solve in 150 ml ddH<sub>2</sub>O<br> |
- | Adjust pH value to 6.5 with HCl to 250 ml | + | Adjust pH value to 6.5 with HCl to 250 ml ddH<sub>2</sub>O.<br> |
<b>sterile filtered</b> | <b>sterile filtered</b> | ||
Line 530: | Line 530: | ||
|<b>20 % Glucose</b> | |<b>20 % Glucose</b> | ||
|Glucose<br> | |Glucose<br> | ||
- | to100 ml | + | to100 ml ddH<sub>2</sub>O<br> |
<b>steril filtered</b> | <b>steril filtered</b> | ||
|20 g<br> <br> <br><br> | |20 g<br> <br> <br><br> | ||
Line 551: | Line 551: | ||
MgCl₂ x 6H₂O<br> | MgCl₂ x 6H₂O<br> | ||
CaCl₂<br> | CaCl₂<br> | ||
- | to 625 ml | + | to 625 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> | <b>autoclaved</b> | ||
|1.25 g<br> | |1.25 g<br> | ||
Line 575: | Line 575: | ||
|Ampicillin<br> | |Ampicillin<br> | ||
Ethanol<br> | Ethanol<br> | ||
- | + | ddH<sub>2</sub>O | |
|1000 mg<br> | |1000 mg<br> | ||
5ml<br> | 5ml<br> | ||
Line 590: | Line 590: | ||
MOPS<br> | MOPS<br> | ||
NaOH<br> | NaOH<br> | ||
- | to 250 ml | + | to 250 ml ddH<sub>2</sub>O<br> |
Adjust pH value to 7.3 with NaOH<br> | Adjust pH value to 7.3 with NaOH<br> | ||
<b>Steril filtered</b> <br><br> | <b>Steril filtered</b> <br><br> | ||
Line 597: | Line 597: | ||
Casamino Acids (Difco)<br> | Casamino Acids (Difco)<br> | ||
Yeast Extract<br> | Yeast Extract<br> | ||
- | to 142.5 ml | + | to 142.5 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> | <b>autoclaved</b> | ||
|<br><br> | |<br><br> | ||
Line 610: | Line 610: | ||
|LB-Medium (Sigma Aldrich)<br> | |LB-Medium (Sigma Aldrich)<br> | ||
Agar<br> | Agar<br> | ||
- | to 400 ml | + | to 400 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> | <b>autoclaved</b> | ||
|10 g | |10 g | ||
Line 619: | Line 619: | ||
|<b> LB-Medium </b> | |<b> LB-Medium </b> | ||
|LB-Medium (Sigma Aldrich)<br> | |LB-Medium (Sigma Aldrich)<br> | ||
- | to 400 ml | + | to 400 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> | <b>autoclaved</b> | ||
|12 g <br> <br> <br> <br> | |12 g <br> <br> <br> <br> | ||
Line 644: | Line 644: | ||
1 M MgSO₄<br> | 1 M MgSO₄<br> | ||
5 M NaCl<br> | 5 M NaCl<br> | ||
- | to 990 ml | + | to 990 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> <br> <br> | <b>autoclaved</b> <br> <br> | ||
<b>Solution 2:</b><br> | <b>Solution 2:</b><br> | ||
Line 664: | Line 664: | ||
Tryptone<br> | Tryptone<br> | ||
Glycerol<br> | Glycerol<br> | ||
- | to 900 ml | + | to 900 ml ddH<sub>2</sub>O<br> |
<b>autoclaved</b> <br> | <b>autoclaved</b> <br> | ||
Add 100 ml 10 x KPP Buffer | Add 100 ml 10 x KPP Buffer | ||
Line 678: | Line 678: | ||
|<b> Xylose Solution (250 mg/ml)</b> | |<b> Xylose Solution (250 mg/ml)</b> | ||
| Xylose<br> | | Xylose<br> | ||
- | to 10 ml | + | to 10 ml ddH<sub>2</sub>O |
|2.5 g <br> <br> <br> | |2.5 g <br> <br> <br> | ||
|- | |- |
Revision as of 11:21, 17 October 2014
Materials
The following section lists the used material in the presented iGEM project:
To read more: click on our contents
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) | GE Healthcare |
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 |
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 |
Sodium Chloride | VWR |
Sodium Hydroxide | Grüssing |
Sodium Phosphate | VWR |
Succrose | Sigma Aldrich |
Temed | Applichem |
Tween 20 | Applichem |
Tetracycline | Serva |
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 |
Na3PO4 Solution (100 mM) | Na3PO4 Solve in 80 ml ddH2O | 1.64 g |
MgSO4 Solution (1 M) | MgSO4 to 100 ml ddH2O | 12.04 g |
Lysis Buffer | Na3PO4 Solution
MgSO4 Solution | 1 ml
1 ml |
PBS-T | 1 x PBS
Tween 20 | 300 µl
300 µl |
Blocking Buffer | BSA
1 x PBS | 0.2 g
40 ml |
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-Mixture | Solution A Solution B | 1.75 ml 1 ml |
8 x CR5-Salts | 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
Strain | Genotype | Reference |
---|---|---|
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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