Berkeley LBL/Laina Notebook
From 2007.igem.org
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crtN | crtN | ||
| - | PCR of crtN | + | PCR of crtN an Heliobacillus mobilis |
| - | G/C content | + | G/C content an annealing region |
crtN-F 40% BglII | crtN-F 40% BglII | ||
Revision as of 05:50, 27 October 2007
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 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)
Results
Sequencing
