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
|
The same concentrations of antibiotics were used for selection of resistant in media.
First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl2. After addition of further 190 ml 100 mM CaCl2 the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage.
For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions.
For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg.
For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop.
By using PCR smallest amount of DNA can be detected and amplified. The principle of PCR is the selective amplification of any region of the DNA. Nevertheless, the sequence at both ends must be known for the binding of two complementary primers. The DNA region of interest is than amplified exponentially while the reproduction of the DNA takes place in three temperature stpes: denaturation of parental DNA at high temperature (95-98 °C), hybridisation of primers (58-65 °C) and DNA elongation (72 °C).
For the amplification a heat-stable DNA polymerase I with a 3’-5’ exonuclease activity (proof reading) is used such as Pfu (from Pyrococcus furiosus) and Phusion (a Pyrocuccus-like enzyme) or the non proof reading enzyme Taq (from Thermus aquaticus).
Phsuion polymerase was used in a 2x master mix adding only primers (20pmol each) and DNA template (~10ng) or alternatively colonies from agar plates (1-5 colonies) in a final volume of 50 µl. For taq and Pfu polymerase following reaction batch was commonly used:
For site directed mutagenesis PCR Pfu turbo polymerase (Stratagene) was used in the same reaction batch described above. The temperature program was as follows:
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