Berkeley LBL/Laina Notebook
From 2007.igem.org
Contents |
Materials
Methods
Primer Design
Biobricking of bch-gene cluster and crtN gene.
Gene:
| bchB | bchE | bchI | bchL | bchM | bchN | [[crtNgene]] |
crtN
PCR of crtN on Heliobacillus mobilis G/C content on annealing region
crtN-F 40% BglII CrtN-R 50% BamHI and XhoI
Primers
crtN-F 5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’ crtN-R 3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’ bchB-F 5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA bchB-R 3'ttaGGATCCccgttcgccttggtttgacttact5' bchE-F ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca bchE-R gctagCTCGAGtattttcatcatgcctctcgt bchI-F ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT bchI-R ttaGGATCCtcggggctgagaaggcgggagca bchL-F ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA bchL-R ttaGGATCCttgggcagaaggtgtggaagca bchM-F ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC bchM-R ttaaGGATCCtctcttcggcttaatttccaacag bchN-F accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac bchN-R attaggatccTCATTCCAGCCACCCCGCTT
PCR
PCR of bch-? genes using - 20ng/ul pHM6 1ul pHM6 1ul dNTP 5ul primer mix 0.5ul enzyme-phusion polymerase 32.5ul H20 50ul total
Digestions
6 ul bch insert 8ul pET29bEBBX 2ul ligase buffer 1ul ligase 3ul H20 20ul total
Dephosphorylation
50 ul pBR3222 6 ul phosphatase buffer 1 ul phosphatase 3 ul H20 Incubate 30" -1hr run gel
Ligations (3hrs @ R.T)
Results
Sequencing
