"
Team:Heidelberg/Notebook/material
From 2008.igem.org
(Difference between revisions)
(→Synthetic oligonucleotides) |
(→Synthetic oligonucleotides) |
||
| Line 701: | Line 701: | ||
===Synthetic oligonucleotides=== | ===Synthetic oligonucleotides=== | ||
| - | All oligonucleotides were purchased from Invitrogen (Karlsruhe) and adjusted to a standard concentration of 100 pmol/µl. | + | All oligonucleotides were purchased from Invitrogen (Karlsruhe) and adjusted to a standard concentration of 100 pmol/<sub>µl</sub>. |
{| class="wikitable" | {| class="wikitable" | ||
Revision as of 17:06, 28 October 2008
Contents |
Material
Buffers & Solution
| SM | 50 mM | Tris, pH 7.5 |
| 20 mM | MgSO4 | |
| 50 mM | NaCl | |
| 0.01 % | gelatine | |
| Tethering buffer, pH 7.0 | 10 mM | potassium phosphate |
| (K2HPO4 : KH2PO4 = 1 :1 ) | ||
| 100 µM | EDTA | |
| 1 µM | L-Methionine | |
| 10 mM | lactic acid | |
| M63 salt | 3 g/l | KH2PO4 |
| 9 g/l | K2HPO4 | |
| 4 g/l | (NH4)2SO4 | |
| 0.5 g/l | FeSO4 | |
| Amino acid mix | 5 g/l | L-threonine |
| 5 g/l | L-histidin | |
| 5 g/l | L-leucine | |
| 5 g/l | L-methionine | |
| M9 salts | 64 g/l | Na2HPO4 x 7H2O |
| 15 g/l | KH2PO4 | |
| 2.5 g/l | NaCl | |
| 5 g/l | NH4Cl |
Kits
| Kit | supplier |
|---|---|
| CompactPrep Plasmid Maxi Kit | Qiagen |
| HISpeed Plasmid Maxi Kit | Qiagen |
| MaxPlax™ Lambda Packaging Extracts | EPICENTRE Biotechnologies |
| QIAEX II Gel Extraction Kit | Qiagen |
| QIAGEN Lambda Mini Kit | Qiagen |
| QIAprep Spin Mini Kit | Qiagen |
| QIAquick Gel Extraction Kit | Qiagen |
| QIAquick PCR Purification Kit | Qiagen |
Marker
| Marker | supplier |
|---|---|
| GeneRuler™ High Range DNA Ladder | MBI Fermentas |
| GeneRuler™ 1kb DNA Ladder Mix | MBI Fermentas |
| GeneRuler™ 1kb Plus DNA Ladder Mix | MBI Fermentas |
Enzymes
| Enzym | supplier |
|---|---|
| Pfu DNA polymerase | Stratagene |
| Pfu turbo DNA polymerase | Stratagene |
| Phusion DNA polymerase | Finnzymes |
| Taq DNA polymerase | MBI Fermentas |
| T4 DNA ligase | MBI Fermentas / New England Biolabs |
| AgeI | New England Biolabs |
| BamHI | New England Biolabs |
| BglI | MBI Fermentas |
| BseJI | MBI Fermentas |
| BspEI | New England Biolabs |
| DpnI | Roche Diagnostics / New England Biolabs |
| DraI | New England Biolabs |
| EcoRI | New England Biolabs |
| EcoRV | New England Biolabs |
| HindIII | New England Biolabs |
| KpnI | New England Biolabs |
| NcoI | New England Biolabs |
| NdeI | New England Biolabs |
| NotI | New England Biolabs |
| PstI | New England Biolabs |
| SacI | New England Biolabs |
| SalI | MBI Fermentas |
| ScaI | New England Biolabs |
| SfcI | New England Biolabs |
| SmiI | MBI Fermentas |
| SpeI | New England Biolabs |
| SpeI | New England Biolabs |
| XbaI | New England Biolabs |
| XhoI | MBI Fermentas |
| XmaI | New England Biolabs |
Plasmidvectors
| Name | application | reference |
|---|---|---|
| BBa_B0015 | terminator | http://partsregistry.org |
| BBa_F1610 | LuxI | http://partsregistry.org |
| BBa_I15030 | AI-1 amplifier | http://partsregistry.org |
| BBa_I20260 | GFP | http://partsregistry.org |
| BBa_J01003 | oriT | http://partsregistry.org |
| BBa_J16002 | cloning vector | http://partsregistry.org |
| BBa_J23107 | constitutive promotor | http://partsregistry.org |
| BBa_T9002 | AI-1 GFP receiver | http://partsregistry.org |
| CheY-mCherry | cloning vector | V. Sourjik, ZMBH |
| ColE1 | colicin E1 | DSMZ |
| ColE9-J | colicin E9 | C. Kleanthous, University of York |
| pBad18 | cloning vector | V. Sourjik, ZMBH |
| pBad33 | cloning vector | V. Sourjik, ZMBH |
| pBluescript II SK (+) | cloning vector | V. Sourjik, ZMBH |
| pDest15 | cloning vector | DKFZ Library |
| pDK4 | visualization | V. Sourjik, ZMBH |
| pDK48 | cloning vector | V. Sourjik, ZMBH |
| pDK58 | cloning vector | V. Sourjik, ZMBH |
| pDK6 | visualization | V. Sourjik, ZMBH |
| pED374 | oriT | K. Derbyshire, Wadsworth |
| pES16 | cloning vector | V. Sourjik, ZMBH |
| pMMB863 | oriT | M. Bagdasarian, MSU |
| pQE-30 | cloning vector | Invitrogen |
| pSB1A2 | cloning vector | http://partsregistry.org |
| pSB2K3 | cloning vector | http://partsregistry.org |
| pTrc99a | cloning vector | V. Sourjik, ZMBH |
| pUB307 | helper plasmid | E. Lanka, BfR |
| RP4 | helper plasmid | M. Bagdasarian, MSU |
Synthetic oligonucleotides
All oligonucleotides were purchased from Invitrogen (Karlsruhe) and adjusted to a standard concentration of 100 pmol/µl.
| Name | SEQUENCE (5´->3´) |
|---|---|
| Bam_fw | GACAAGTGTTGGCCATGGAACAGG |
| Bam_rv | GCCGTCTGTGATGGCTTCCATG |
| cI_mut_fw | GCGTCTGGGTGGTGATGAGTTCACCTTCAAAAAACTG |
| cI_mut_rv | CAGTTTTTTGAAGGTGAACTCATCACCACCCAGACGC |
| CmR_EcoRI_mut_fw | GAATGCTCATCCGGAGTTCCGTATGGCAATG |
| CmR_EcoRI_mut_rev | CATTGCCATACGGAACTCCGGATGAGCATTC |
| CmR_fw | GCTAAAATGGAGAAAAAAATCACTGG |
| CmR_new_fw | TACGAGGTACCTTTACAGCTAGCTCAGTCCTAGGTATTATGC |
| CmR_new_rev | TATATAAGCTTTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTG |
| CmR_Prefix_fw | GAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGG |
| CmR_rv | AGGTTCTCCTTTATTAGCCGGATCCTCTAGATTACGCC |
| CmR_Suffix_rv | CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGCCCACCC |
| colE1_kil_prot_rv_A_SpeI | TATATACTAGTACTACTGAACCGCGATCCCCG |
| colE1_mut_EcoRI_fw | GGTATTGCTATTGTTACAGGTATTCTATGCTCCTATATTGATAAG |
| colE1_mut_EcoRI_rv | CTTATCAATATAGGAGCATAGAATACCTGTAACAATAGCAATACC |
| colE1_mut_PstI_1_fw | GCAGTAAAAGTGAAAGTTCAGCAGCTATTCATGCAACTGC |
| colE1_mut_PstI_1_rv | GCAGTTGCATGAATAGCTGCTGAACTTTCACTTTTACTGC |
| colE1_mut_PstI_2_fw | GCTGCCCGGGCAAAAGCAGCAGCGGAAGCACAGG |
| colE1_mut_PstI_2_rv | CCTGTGCTTCCGCTGCTGCTTTTGCCCGGGCAGC |
| colE1_mut_PstI_3_fw | CATTAGAGAAGAAAGCAGCAGATGCAGGGGTGAG |
| colE1_mut_PstI_3_rv | CTCACCCCTGCATCTGCTGCTTTCTTCTCTAATG |
| colE1_prot_fw_BamH1 | TACGAGGATCCATGGAAACCGCGGTAGCG |
| colE1_prot_rv_HindIII | TATATAAGCTTTTAAATCCCTAACACCTC |
| colE9_lysProt_rv_A_SpeI | TATATACTAGTACTAGGTTTTCGGCTTAGAACCCC |
| colE9_mut_EcoRI_fw | GTTGGGTGGACGATTCGAGAGTTCAATGGGGAAATAAAAATG |
| colE9_mut_EcoRI_rv | CATTTTTATTTCCCCATTGAACTCTCGAATCGTCCACCCAAC |
| colE9_plasmid_rv_A_SpeI | TATATACTAGTACACATGGAACTTTTGTGAC |
| colE9_prot_fw_BamH1 | TACGAGGATCCATCGATTTGCCCATGACCC |
| colE9_prot_rv_XmaI | TATATCCCGGGTTACTTACCTCGGTGAATATCG |
| DK13 | TCACCCGCACGCGC |
| DK167 | AGGATACTAGTAGGCCATTACTTT |
| DK9b | CGATGCGGCCGCTCAAAATGTTTCCCAGTTTGG |
| F1610_fw_XbaI | TACGATCTAGAAAAGAGGAGAAATACTAG |
| F1610_rv_HindIII | TATATAAGCTTTATAAACGCAGAAAGGCCC |
| GAM_fw | AGTGCTTTAGCGTTAACTTCCG |
| GAM_rv | GGTTTTACCGCATACCAATAACG |
| GFP_CmR_fw | CTCGTTGGTACCTCTAGATTTACAGCTAGCTCAGTCCTAGG |
| GFP_CmR_rv | TATTCGACCGGTACTAGTTATAAACGCAGAAAGGCCCACC |
| GFP_new_fw | TACGAGAGCTCTTTACAGCTAGCTCAGTCCTAGG |
| GFP_new_rv | TATATACCGGTACTAGTTATAAACGCAGAAAGGCCCACC |
| Lambda_insert_fw | TTGTAAAAACAGCCCTCCTC |
| Lambda_insert_rv | GATATGACTATCAAGGCCGC |
| LuxP_mut_F | CGTGAATTAGCAACAGAGTTCGGAAAGTTCTTCCC |
| LuxP_mut_R | GGGAAGAACTTTCCGAACTCTGTTGCTAATTCACG |
| LuxP_prefix_F | GAGGGAGAATTCGCGGCCGCTTCTAGATGAAGAAAGCGTTACTATTTTC |
| LuxP_sufffix_R | GGAGAGCTGCAGCGGCCGCTACTAGTAATTATCTGAATATCTAAATGCG |
| LuxPc | ATTACGCGGCCGCAGGAAACAGACCATGAAGAAAGCGTTACTATTTTCCC |
| LuxPd | GTAATGTCGACTCAATTATCTGAATATC |
| LuxP-seq-FW | CCCGTCCTGCCAGTGAGC |
| LuxQa | ATCGACCATGGGCAATAAATTTCGCTTAGC |
| LuxQc | GTAATGGATCCTTAGTGGAGGCTTGAGCC |
| LuxQTar_1a | CACAAATCATTGCCAATGAACGTATGTTGCTTACTCCGCTGG |
| LuxQTar_1b | CCAGCGGAGTAAGCAACATACGTTCATTGGCAATGATTTGTG |
| LuxQTar_2a | CTTAGCGACCATGAGCCATGAGTTTGCCCAGTGGCAACTGGC |
| LuxQTar_2b | GCCAGTTGCCACTGGGCAAACTCATGGCTCATGGTCGCTAAG |
| LuxS mutBbaIR | CCACACCCACTTCTAGGATGTTCTTCGCGATTTGC |
| LuxS mutXbaIF | GCAAATCGCGAAGAACATCCTAGAAGTGGGTGTGG |
| LuxS_mut_fw | CATCCTTTCTGAGAAAGGCATTCATACATTAGAGC |
| LuxS_mut_rev | GCTCTAATGTATGAATGCCTTTCTCAGAAAGGATG |
| LuxS_prefix_F | GGAGAGGAATTCGCGGCCGCTTCTAGATGGGCAATGCACCAGCGGTTCG |
| luxS_suffix_R | GAGGGACTGCAGCGGCCGCTACTAGTAGTCGATGCGTAGCTCTCTCAGC |
| LuxSa | ATCAGTCCATGGGCAATGCACCAGCGGTTCG |
| LuxSb | GTAATGGATCCTTAGTCGATGCGTAGC |
| mut_insert_fw | CTGAGGGGACGGTACCTCTACATTTACAGCTAGCTCAG |
| mut_insert_rv | CTGAGCTAGCTGTAAATGTAGAGGTACCGTCCCCTCAG |
| mut_insert2_fw | GGCAGGCGGGGCGTAATCTATAGGATCCGGCTAATAAAGG |
| mut_insert2_rv | CCTTTATTAGCCGGATCCTATAGATTACGCCCCGCCTGCC |
| mut_kpn1_pBlue | CGAGGGGGGGCCCGGTTCCCAATTCGCCCTATAG |
| mut_kpn1_pBlue | CTATAGGGCGAATTGGGAACCGGGCCCCCCCTCG |
| oriT_pre | GAATTCGCGGCCGCTTCTAGAGGACAGGCTCATGCCGGCCGC |
| oriT_RP4_fw | CTCGTTTCTAGAACTAGTGACAGGCTCATGCCGGCCGC |
| oriT_RP4_rv | TATTCGGGTACCGTCCCCTCAGTTCAGTAATTTCCTGC |
| oriT_suf | CTGCAGCGGCCGCTACTAGTAGTCCCCTCAGTTCAGTAATTTCCTGC |
| T9002_Lux_pR_rv_SpeI_BamHI_RBS | TATATACTAGTGGATCCGGTTCTGTTTCCTCTCTAGTATTTATTCGAC |
| T9002_LuxpR_Not_Eco_Xba_G_fw | TACGAGAATTCGCGGCCGCTTCTAGAGTCCCTATCAGTGATAGAGATTG |
| Term_new_fw | TACGAAAGCTTCCAGGCATCAAATAAAACGAAAGG |
| Term_new_rv | TATATGAGCTCTATAAACGCAGAAAGGCCCACCC |
| VF2 | TGCCACCTGACGTCTAAGAA |
| VIC121 | TTTATCGCAACTCTCTACTG |
| VIC122 | CTGATTTAATCTGTATCAGG |
| VIC131 | ATGTGTGGAATTGTGAGCGG |
| VIC132 | CTGATTTAATCTGTATCAGG |
| VR | ATTACCGCCTTTGAGTGAGC |
Phages
| Name | application | reference |
|---|---|---|
| Lambda cI mut | cI deleted | W. Reiser, ZMBH |
| Lambda cI857 | heat inducible | MBI Fermentas |
Bacteria
| E.coli strain | usage | reference |
|---|---|---|
| DH5a | amplification of plasmids | Invitrogen |
| HCB33 | swarm assays | V. Sourjik, ZMBH |
| MG1655 | swarm assays | V. Sourjik, ZMBH |
| TOP10 | amplification of plasmids | Invitrogen |
| UU1250 | chemotaxis receptor knock out | V. Sourjik, ZMBH |
Bacteria Growth Media
| E.coli strain | usage | reference |
|---|---|---|
| DH5a | amplification of plasmids | Invitrogen |
| HCB33 | swarm assays | V. Sourjik, ZMBH |
| MG1655 | swarm assays | V. Sourjik, ZMBH |
| TOP10 | amplification of plasmids | Invitrogen |
| UU1250 | chemotaxis receptor knock out | V. Sourjik, ZMBH |
For cultivation of phages the media were supplied with 0.2 % maltose and 10 mM MgSO4. For preparation of agar plates 15 g/l agar, for preparation of top agar 7 g/l agar was added prior the autoclaving. For selection of resistant bacteria following antibiotics were added during cooling of at agar at about 50 °C:
| Antibiotic | concentration |
|---|---|
| Ampicillin | 100 µg/ml |
| Chloramphenicol | 35 µg/ml |
| Kanamycin | 50 µg/ml |
The same concentrations of antibiotics were used for selection of resistant in media.
Methods
DNA amplification using polymerase chain reaction (PCR)
| 1 µl template (10 ng) / colonies |
| 2 µl primer 1 (10 pmol/µl) |
| 2 µl primer 2 (10 pmol/µl) |
| 1 µl polymerase (2,5 Units) |
| 1 µl dNTP mix (10 mM) |
| 5 µl 10x buffer |
| 38 µl dH2O |
The PCR procedure was as follows:
| Initiale denaturation | 95- 98 °C, 3-5 min | 1 cycle |
| denaturation | 95-98 °C, 15 sec - 1 min | |
| annealing | 58-65 °C, 15 sec - 1 min | 25-28 cycles |
| elongation | 72 °C, 15 sec - 3 min | |
| termination | 72 °C, 5 – 10 min | 1 cycle |
| 4 °C | forever | |
For site directed mutagenesis PCR Pfu turbo polymerase (Stratagene) was used in the same reaction batch described above. The temperature program was as follows:
| initiale denaturation | 95 °C, 30 sec | 1 cycle |
| denaturation | 95 °C, 30 sec | |
| annealing | 55 °C, 1 min | 16 cycles |
| elongation | 68 °C, 2 min per kb of plasmid | |
| termination | 68 °C, 10 min | 1 cycle |
