Berkeley LBL/Laina Notebook
From 2007.igem.org
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'''Digestions''' | '''Digestions''' | ||
| + | 6 ul bch insert | ||
| + | 8ul pET29bEBBX | ||
| + | 2ul ligase buffer | ||
| + | 1ul ligase | ||
| + | 3ul H20 | ||
| + | 20ul total | ||
'''Dephosphorylation''' | '''Dephosphorylation''' | ||
| Line 52: | Line 58: | ||
Incubate 30" -1hr | Incubate 30" -1hr | ||
run gel | run gel | ||
| + | |||
| + | Ligations (3hrs @ R.T) | ||
| + | |||
== '''Results''' == | == '''Results''' == | ||
Revision as of 05:27, 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 on Heliobacillus mobilis G/C content on annealing region
crtN-F 40% BglII CrtN-R 50% BamHI and XhoI
Primers
3crtN-F 3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’
crtN-R 5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
bch B 3'ttaGGATCCccgttcgccttggtttgacttact5'
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
