Klonování genu pro glukosa oxidasu z A. niger >AF234246.2:486-2303 Aspergillus niger glucose oxidase (GO2) gene, complete cds ATGCAGACTCTCCTTGTGAGCTCGCTTGTGGTCTCCCTCGCTGCGGCCCTGCCACACTACATCAGGAGCA ATGGCATTGAAGCCAGCCTCCTGACTGATCCCAAGGATGTCTCCGGCCGCACGGTCGACTACATCATCGC TGGTGGAGGTCTGACTGGACTCACCACCGCTGCTCGTCTGACGGAGAACCCCAACATCAGTGTGCTCGTC ATCGAAAGTGGCTCCTACGAGTCGGACAGAGGTCCTATCATTGAGGACCTGAACGCCTACGGCGACATCT TTGGCAGCAGTGTAGACCACGCCTACGAGACCGTGGAGCTCGCTACCAACAATCAAACCGCGCTGATCCG CTCCGGAAATGGTCTCGGTGGCTCTACTCTAGTGAATGGTGGCACCTGGACTCGCCCCCACAAGGCACAG GTTGACTCTTGGGAGACTGTCTTTGGAAATGAGGGCTGGAACTGGGACAATGTGGCCGCCTACTCCCTCC AGGCTGAGCGTGCTCGCGCACCAAATGCCAAACAGATCGCTGCTGGCCACTACTTCAACGCATCCTGCCA TGGTGTTAATGGTACTGTCCATGCCGGACCCCGCGACACCGGCGATGACTATTCTCCCATCGTCAAGGCT CTCATGAGCGCTGTCGAAGACCGGGGCGTTCCCACCAAGAAAGACTTCGGATGCGGTGACCCCCATGGTG TGTCCATGTTCCCCAACACCTTGCACGAAGACCAAGTGCGCTCCGATGCCGCTCGCGAATGGCTACTTCC CAACTACCAACGTCCCAACCTGCAAGTCCTGACCGGACAGTATGTTGGTAAGGTGCTCCTTAGCCAGAAC GGCACCACCCCTCGTGCCGTTGGCGTGGAATTCGGCACCCACAAGGGCAACACCCACAACGTTTACGCTA AGCACGAGGTCCTCCTGGCCGCGGGCTCCGCTGTCTCTCCCACAATCCTCGAATATTCCGGTATCGGAAT GAAGTCCATCCTGGAGCCCCTTGGTATCGACACCGTCGTTGACCTGCCCGTCGGCTTGAACCTGCAGGAC CAGACCACCGCTACCGTCCGCTCCCGCATCACCTCTGCTGGTGCAGGACAGGGACAGGCCGCTTGGTTCG CCACCTTCAACGAGACCTTTGGTGACTATTCCGAAAAGGCACACGAGCTGCTCAACACCAAGCTGGAGCA GTGGGCCGAAGAGGCCGTCGCCCGTGGCGGATTCCACAACACCACCGCCTTGCTCATCCAGTACGAGAAC TACCGCGACTGGATTGTCAATCACAACGTCGCGTACTCGGAACTCTTCCTCGACACTGCCGGAGTGGCCA GCTTCGATGTGTGGGACCTTCTGCCCTTCGACCGAGGATACGTCCACATCCTCGACAAGGACCCCTACCT CCACCACTTTGCCTACGACCCTCAGTACTTCCTCAACGAGCTCGACCTGCTCGGTCAGGCTGCCGCTACT CAGCTGGCCCGCAACATCTCCAACTCCGGTGCCATGCAGACCTACTTCGCTGGGGAGATACTCCCCGGTG ATAACCTCGCGTATGATGCCGATTTGAGCGCCTGGACTGAGTACATCCCGTACCACTTCCGTCCTAACTA CCATGACGTAGGTACTTGCTCCATGATGCCGAAGGAGATGGGCAGTGTTGTTGATAATGCTGCCCGTGTG TATGGTGTGCGGGGACTGCGTGTCATTGATGGTTCTATTCCTCCTACGCAAATGTCGTCCCATGTCATGA CGGTGTTCTATGCCATGGCTTTGAAAATTTCGGATGCTATCTTGGAAGATTATGCTTCCATGCAGTGA N-HIS,T7-GO2-HIS BamH1(GGATCC) XhoI(CTCGAG) 5` TCGCGGATCCCTGCCACACTACATCAGGAGCA 5` GGTGCTCGAGCTGCATGGAAGCATAATCTTCCA N-HIS,T7-GO2 BamH1(GGATCC) XhoI(CTCGAG) 5` TCGCGGATCCCTGCCACACTACATCAGGAGCA 5` GGTGCTCGAGTCACTGCATGGAAGCATAATCTTC N-HIS-GO2 NdeI(CATATG) XhoI(CTCGAG) 5` TATGCATATGCTGCCACACTACATCAGGAGCA 5` GGTGCTCGAGTCACTGCATGGAAGCATAATCTTC >AAF59929.2 glucose oxidase [Aspergillus niger] MQTLLVSSLVVSLAAALPHYIRSNGIEASLLTDPKDVSGRTVDYIIAGGGLTGLTTAARLTENPNISVLV IESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQ VDSWETVFGNEGWNWDNVAAYSLQAERARAPNAKQIAAGHYFNASCHGVNGTVHAGPRDTGDDYSPIVKA LMSAVEDRGVPTKKDFGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQN GTTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQD QTTATVRSRITSAGAGQGQAAWFATFNETFGDYSEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYEN YRDWIVNHNVAYSELFLDTAGVASFDVWDLLPFDRGYVHILDKDPYLHHFAYDPQYFLNELDLLGQAAAT QLARNISNSGAMQTYFAGEILPGDNLAYDADLSAWTEYIPYHFRPNYHDVGTCSMMPKEMGSVVDNAARV YGVRGLRVIDGSIPPTQMSSHVMTVFYAMALKISDAILEDYASMQ Amplifikace genu GO2 z gDNA Aspergillus niger Materiál: Elizyme HS ROBUST MIX RED (Elisabeth Pharmacon) Primery (viz. přiložený Datasheet): N-HIS,T7-GO2-HIS (T7-GO2-HIS_FW/GO2-HIS_REV) N-HIS,T7-GO2 (T7-GO2-HIS_FW/GO2_REV) N-HIS-GO2 (HIS-GO2_FW/GO2_REV) Složení PCR reakční směsi (50 ul) HS ROBUST MIX RED 25.0 ul FW primer (10 uM) 2.0 ul REV primer (10 uM) 2.0 ul PCR voda 16.0 ul gDNA 5.0 ul Teplotní protokol: 95°C 2:30 1 x 95°C 10s 60°C 20s 40 x 72°C 50s 72°C 5 min 1 x Obsah obrázku text Popis byl vytvořen automaticky Přečištění PCR produktu Materiál: Monarch® PCR & DNA Cleanup Kit (5 μg) (https://international.neb.com/-/media/nebus/files/protocols/t1030_quick_protocol_card_monarch_pcrd na_cleanup.pdf?rev=a562d8f1f21741b0ac6d59ea9020cef3&hash=E4845F80E8B2BE925A09DAD6562A41EC Restrikční analýza Materiál: Restriktázy NdeI (https://international.neb.com/products/r0111-ndei#Product%20Information) XhoI (https://international.neb.com/products/r0146-xhoi#Product%20Information) BamHI-HF (https://international.neb.com/products/r3136-bamhi-hf#Product%20Information) Vektor pET28a Složení směsi RA (50 ul) CutSmart buffer (10x) 5.0 ul PCR produkt/vektor 15 ul PCR voda 28 ul NcoI-HF/ BamHI-HF 1.0 ul (10U) XhoI 1.0 ul (10U) Teplotní protokol: Inkubace při 37°C po dobu 3 hodin Izolace naštěpeného PCR produktu a plasmidu z agarózového gelu Materiál: Monarch® DNA Gel Extraction Kit (https://international.neb.com/-/media/nebus/files/protocols/t1020_quick_protocol_card_monarch_dna_ gel_extraction.pdf?rev=09308c01500f43c6a8589b01845765d9&hash=0FD4350A9DE2AEAC84913DB2A00140AF) Ligace PCR produktu do vektoru pET28a Materiál: Instant Sticky-end Ligase Master Mix (https://international.neb.com/protocols/2012/08/27/protocol-transfer-master-mix-to-ice-prior-to-re action-set-up-mix-tube-by-finger-flicking-before-u) Protokol: 1. Transfer master mix to ice prior to reaction set up. Mix tube by finger flicking before use. 2. Combine 100 ng of vector pET28a with a 3-fold molar excess of insert and adjust volume to 5 μl with dH2O. 3. Add 5 μl of Instant Sticky-end Ligase Master Mix, mix thoroughly by pipetting up and down 7-10 times, and place on ice. The sample is now ready to be used for transformation. Transformace vektoru po ligaci do NEB 5-alpha E. coli Materiál: NEB 5-alpha Competent E. coli (High Efficiency) (https://international.neb.com/products/c2987-neb-5-alpha-competent-e-coli-high-efficiency#Product% 20Information) Protokol: 1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes. 2. Add 2 µl of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. 3. Place the mixture on ice for 30 minutes. Do not mix. 4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. 5. Place on ice for 5 minutes. Do not mix. 6. Pipette 950 µl of room temperature SOC into the mixture. 7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. 8. Warm selection plates to 37°C. 9. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.