Berkeley LBL/Laina Notebook

From 2007.igem.org

(Difference between revisions)
('''Methods''')
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'''Digestions'''
'''Digestions'''
 +
  6 ul bch insert
 +
  8ul pET29bEBBX
 +
  2ul ligase buffer
 +
  1ul ligase
 +
  3ul H20
 +
  20ul total
'''Dephosphorylation'''
'''Dephosphorylation'''
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   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

Discussion