Team:Saarland/Materials

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Latest revision as of 12:22, 17 October 2014



Materials


The following section lists the used material in the presented iGEM project:




Skip to a chapter by clicking the corresponding marking above

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
to 100 ml ddH2O
autoclaved

2,71 g

12.5 g


Stacking Gel Buffer Tris

SDS
Adjust pH value to 6.7 to 100 ml ddH2O

6.05 g

0.4 g

Stacking Gel (4 %) ddH2O

Stacking Gel Buffer
Rotiphorese Gel 30
Temed
10 % APS

1193 µl

500 µl
267 µl
5 µl
10 µl

Separating Gel Buffer Tris

SDS
Adjust pH value to 8.8 To 100 ml ddH2O

18.17 g

0.4 g

Separating Gel (12 %) ddH2O

Separating Gel Buffer
Rotiphorese Gel 30
Temed
10 % APS

1350 µl

1000 µl
1600 µl
10 µl
20 µl

5 x SDS Sample Buffer Tris

2-ME
SDS
Glycerol
Bromophenol Blue
Adjust pH value to 6.5 with HCl to 50 ml ddH2O

1.21 g

3.487 g
5 g
10 ml
25 mg

Na3PO4 Solution (100 mM) Na3PO4

Solve in 80 ml ddH2O
Adjust pH value to 6.5 with H3PO4 to 100 ml ddH2O

1.64 g



MgSO4 Solution (1 M) MgSO4

to 100 ml ddH2O

12.04 g


Lysis Buffer Na3PO4 Solution

MgSO4 Solution
add 5 mg Lysozyme per ml Lysis Buffer before use

1 ml

1 ml

PBS-T 1 x PBS

Tween 20

300 µl

300 µl

Blocking Buffer BSA

1 x PBS
Tween 20

0.2 g

40 ml
40 µl

10 x TBE Trizma-Baze

Boric Acid
EDTA
to 1000 ml ddH2O

162 g

27.5 g
9.3 g

12 % Proline Proline

to 25 ml ddH2O
sterile filtered

3 g



2 x AB3 Antibiotic Medium NO. 3 (Difco)

to 200 ml ddH2O
autoclaved

7 g



2 x SMM Maleic Acid

NaOH
MgCl₂ x 6H2O
Succrose

Solve in 150 ml ddH2O
Adjust pH value to 6.5 with HCl to 250 ml ddH2O.

sterile filtered

1.16 g

0,8 g
2.03 g
85.58 g





20 % Glucose Glucose

to100 ml ddH2O
steril filtered

20 g



3.5 ml CR5-Topagar-Mixture Solution A

Solution B
8 x CR5-Salts
12 % Proline
20 % Glucose

1.75 ml

1 ml
0.346 ml
0.175 ml
0.175 ml

8 x CR5-Salts K₂SO₄

KH₂PO₄
MgCl₂ x 6H₂O
CaCl₂
to 625 ml ddH2O
autoclaved

1.25 g

0.25 g
50 g
11 g


Loading Dye for Agarose Gels 25 mM Tris-HCl

Adjust pH value to 7.8 with HCl
Bromophenol Blue
Xylene Cyanol
Glycerol
EDTA

4 ml


3 mg
3 mg
6 ml

Ampicillin Stock Solution (100 mg/ml) Ampicillin

Ethanol
ddH2O

1000 mg

5ml
5ml

Chloramphenicol Stock Solution (10 mg/ml) Chloramphenicol

to 10 ml Ethanol

100mg

CR5-Topagar SolutionA:

Succrose
MOPS
NaOH
to 250 ml ddH2O
Adjust pH value to 7.3 with NaOH
Steril filtered

Solution B:
Agar
Casamino Acids (Difco)
Yeast Extract
to 142.5 ml ddH2O
autoclaved



51.5 g
3.25 g
0.3 g





2 g
0.1 g
5 g


LB-Agar LB-Medium (Sigma Aldrich)

Agar
to 400 ml ddH2O
autoclaved

10 g

6 g


LB-Medium LB-Medium (Sigma Aldrich)

to 400 ml ddH2O
autoclaved

12 g



PEG-P PEG-6000

1 x SMM
autoclaved

20 g

50 ml

SMMP 2 x AB3

2 x SMM

25ml

25 ml

SOC-Medium Solution 1:

Bacto Tryptone
Yeast Extrakt
1 M KCl
1 M MgCl₂
1 M MgSO₄
5 M NaCl
to 990 ml ddH2O
autoclaved

Solution 2:
40 % Glucose (w/v)
sterile filtered

Add Solution 1 to Solution 2


20 g
5 g
2,5 ml
10 ml
10 ml
2 ml



10 ml




TB-Medium Yeast Extrakt

Tryptone
Glycerol
to 900 ml ddH2O
autoclaved
Add 100 ml 10 x KPP Buffer

24 g

12 g
4 ml



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
50 ml

Sarstedt
Electrophoresis systems:

PowerPACTM High-Current Power Supply
Mini PROTEANTM 3 Cell
PerfectBlue Gel System Mini S


Biorad
Biorad
Peqlab

Western Blot System:

Blotting Device: OwlTM Separation System
Power Supply: Voltcraft DPS-4005PFC


Thermo Fisher Scientific
Conrad Electronic

Fluorescence microscopy:

Inverse Fluorescence microscope Axiovert 200M
CCD Digital camera Coolsnap HQ2


Zeiss
Photometrics

Scale (MXX2001) Denver Instruments
Micro scale (BP 210 S) Sartorius
Gloves:

Nitrile
Latex

VWR
Imaging System Chemi DOC XRS+ Biorad
Incubator: Innova 4230 Innova
Cuvettes:

Polystyrol/Polystyrene 10 x 4x 45 mm
Quarz

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
2 ml
PCR-Softtubes

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
Multifuge 1L

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)
7697 galU galK rpsL endA1 nupG

Invitrogen
B. megaterium MS941 ΔnprM Wittchen et al., 1995



8. Antibodies

Antibody Manufacturer
Anti-GFP, N-terminal antibody produced in rabbit Sigma Aldrich
Anti-Rabbit IgG peroxidase produced in goat Sigma Aldrich





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