Berkeley LBL/Laina Notebook
From 2007.igem.org
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Plasmids, Cells, Miniprep Kits, IPTG, Antibiotics. | Plasmids, Cells, Miniprep Kits, IPTG, Antibiotics. | ||
Revision as of 06:09, 27 October 2007
Contents |
Introduction
Materials
Plasmids, Cells, Miniprep Kits, IPTG, Antibiotics.
Methods
Primer Design
Biobricking of bch-gene cluster and crtN gene.
Plasmids:
| pET29EBBX|pBR322|pHM6
Genes:
crtN PCR of crtN an Heliobacillus mobilis G/C content an annealing region crtN-F 40% BglII CrtN-R 50% BamHI and XhoI Primers crtN-F 5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’ crtN-R 3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’ bchB-F 5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA3' bchB-R 3'ttaGGATCCccgttcgccttggtttgacttact5' bchE-F 5'ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca3' bchE-R 3'gctagCTCGAGtattttcatcatgcctctcgt5' bchI-F 5'ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT3' bchI-R 3'ttaGGATCCtcggggctgagaaggcgggagca5' bchL-F 5'ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA3' bchL-R 3'ttaGGATCCttgggcagaaggtgtggaagca5' bchM-F 5'ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC3' bchM-R 3'ttaaGGATCCtctcttcggcttaatttccaacag5' bchN-F 5'accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac3' bchN-R 3'attaggatccTCATTCCAGCCACCCCGCTT5' 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) ResultsSequencing Discussion |
