From 2007.igem.org

Contents 1 Materials 2 Methods 3 Results 4 Discussion

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

Discussion