Team:Saarland/Test
From 2014.igem.org
(Difference between revisions)
Line 347: | Line 347: | ||
!Quantity | !Quantity | ||
|- | |- | ||
- | |10 x KPP Buffer | + | |<b>10 x KPP Buffer</b> |
|KH₂PO₄<br> | |KH₂PO₄<br> | ||
K₂HPO₄<br> | K₂HPO₄<br> | ||
to 100 ml ddH2O | to 100 ml ddH2O | ||
- | autoclaved | + | <b>autoclaved</b> |
|2,71 g<br> | |2,71 g<br> | ||
12.5 g <br> <br> | 12.5 g <br> <br> | ||
|- | |- | ||
- | |Stacking Gel Buffer | + | |<b>Stacking Gel Buffer</b> |
|Tris<br> | |Tris<br> | ||
SDS<br> | SDS<br> | ||
Line 364: | Line 364: | ||
|- | |- | ||
- | |Stacking Gel (4 %) | + | |<b>Stacking Gel (4 %)</b> |
|ddH2O<br> | |ddH2O<br> | ||
Stacking Gel Buffer<br> | Stacking Gel Buffer<br> | ||
Line 376: | Line 376: | ||
10 µl<br> | 10 µl<br> | ||
|- | |- | ||
- | |Separating Gel Buffer | + | |<b>Separating Gel Buffer</b> |
|Tris<br> | |Tris<br> | ||
SDS<br> | SDS<br> | ||
Line 385: | Line 385: | ||
<br> | <br> | ||
|- | |- | ||
- | |Separating Gel (12%) | + | |<b>Separating Gel (12%)</b> |
|ddH2O<br> | |ddH2O<br> | ||
Separating Gel Buffer<br> | Separating Gel Buffer<br> | ||
Line 397: | Line 397: | ||
20 µl<br> | 20 µl<br> | ||
|- | |- | ||
- | |5 x SDS Sample Buffer | + | |<b>5 x SDS Sample Buffer</b> |
|Tris <br> | |Tris <br> | ||
2-ME<br> | 2-ME<br> | ||
Line 411: | Line 411: | ||
25 mg<br> <br> | 25 mg<br> <br> | ||
|- | |- | ||
- | |10 x TBE | + | |<b>10 x TBE</b> |
|Trizma-Baze<br> | |Trizma-Baze<br> | ||
Boric Acid<br> | Boric Acid<br> | ||
Line 420: | Line 420: | ||
9.3 g<br><br> | 9.3 g<br><br> | ||
|- | |- | ||
- | |12 % Proline | + | |<b>12 % Proline</b> |
|Proline<br> | |Proline<br> | ||
to 25 ml ddH2O | to 25 ml ddH2O | ||
- | sterile filtered | + | <b>sterile filtered</b> |
- | |3 g <br> <br> | + | |3 g <br> <br><br> |
|- | |- | ||
+ | | | ||
Revision as of 08:48, 9 October 2014
Materials
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 sterile filtered | 3 g |
|