Berkeley LBL/Laina Notebook

Revision as of 05:27, 27 October 2007 by Silvapy (Talk | contribs)

Materials

Primer Design

Biobricking of bch-gene cluster and crtN gene.

Gene:

[[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)

Sequencing