Team:Saarland/Test

From 2014.igem.org

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<h1>Materials</h1>
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<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
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-
| New England Biolabs
+
-
|-
+
-
| 2-Mercapto Ethanol
+
-
| Merck
+
-
|-
+
-
| Acetic Acid 100%
+
-
| zChL UdS
+
-
|-
+
-
| Agar Kobe I
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| Roth
+
-
|-
+
-
| Agarose
+
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| Peqlab, Biozym
+
-
|-
+
-
| Ampicillin 
+
-
| Roth
+
-
|-
+
-
| APS (Ammoniumpersulfat)
+
-
| zChL UdS
+
-
|-
+
-
| BactoTM Casamino-acids
+
-
| BD
+
-
|-
+
-
| BactoTM Tryptone
+
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| BD
+
-
|-
+
-
| BactoTM Yeast-Extract
+
-
| BD
+
-
|-
+
-
| Boric Acid
+
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| Merck
+
-
|-
+
-
| Bovine Serum Albumin (BSA)
+
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| 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
+
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| zChL UdS
+
-
|-
+
-
| Hydrochloric Acid 37 % (HCl)
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-
| VWR
+
-
|-
+
-
| Immersion Oil
+
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| 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>
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-
<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> <br>
+
-
|-
+
-
|<b> PEG-P </b>
+
-
|PEG-6000<br>
+
-
1 x SMM<br>
+
-
<b>autoclaved</b>
+
-
|20 g<br>
+
-
50 ml<br><br>
+
-
|-
+
-
|<b>SMMP</b>
+
-
|2 x AB3<br>
+
-
2 x SMM
+
-
|25ml<br>
+
-
25 ml<br>
+
-
|-
+
-
|<b> SOC-Medium </b>
+
-
|<b>Solution 1:</b> <br>
+
-
Bacto Tryptone<br>
+
-
Yeast Extrakt<br>
+
-
1 M KCl<br>
+
-
1 M MgCl₂<br>
+
-
1 M  MgSO₄<br>
+
-
5 M NaCl<br>
+
-
to 990 ml ddH2O<br>
+
-
<b>autoclaved</b> <br> <br>
+
-
<b>Solution 2:</b><br>
+
-
40 % Glucose (w/v)<br>
+
-
<b>sterile filtered</b><br> <br>
+
-
Add Solution 1 to Solution 2
+
-
| <br>
+
-
20 g<br>
+
-
5 g<br>
+
-
2,5 ml<br>
+
-
10 ml<br>
+
-
10 ml<br>
+
-
2 ml<br> <br><br> <br>
+
-
10 ml<br>
+
-
<br> <br> <br> <br>
+
-
|-
+
-
| <b> TB-Medium </b>
+
-
|Yeast Extrakt<br>
+
-
Tryptone<br>
+
-
Glycerol<br>
+
-
to 900 ml ddH2O<br>
+
-
<b>autoclaved</b> <br>
+
-
Add 100 ml 10 x KPP Buffer
+
-
|24 g<br>
+
-
12 g<br>
+
-
4 ml<br> <br> <br> <br>
+
-
|-
+
-
|<b> Tetracyclin Stammlösung (10 mg/ml) </b>
+
-
|Tetracycline<br>
+
-
to 10 ml Ethanol
+
-
|100 mg <br> <br> <br>
+
-
|-
+
-
|<b> Xylose Solution (250 mg/ml)</b>
+
-
| Xylose<br>
+
-
to 10 ml ddH2O
+
-
|2.5 g <br> <br> <br>
+
-
|-
+
-
|}
+
-
<br>
+
-
 
+
-
<h2> 5. Equipment </h2>
+
-
<br>
+
-
{|
+
-
|+
+
-
!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
+
-
|-
+
-
|<b>Falcon Tubes:</b> <br>
+
-
15 ml <br>
+
-
50 ml<br>
+
-
|Sarstedt
+
-
|-
+
-
|<b>Electrophoresis systems:</b> <br>
+
-
PowerPACTM High-Current Power Supply<br>
+
-
Mini PROTEANTM 3 Cell<br>
+
-
PerfectBlue Gel System Mini S
+
-
|<br>
+
-
Biorad<br>
+
-
Biorad<br>
+
-
Peqlab<br>
+
-
|-
+
-
| <b>Western Blot System:</b> <br>
+
-
Blotting Device: OwlTM Separation System<br>
+
-
Power Supply: Voltcraft DPS-4005PFC
+
-
| <br>
+
-
Thermo Fisher Scientific<br>
+
-
Conrad Electronic<br>
+
-
|-
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
|}
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
</div>
</div>
</div>
</div>
 +
 +
{{Team:Saarland/footer}}

Latest revision as of 12:39, 17 October 2014


Retrieved from "http://2014.igem.org/Team:Saarland/Test"