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Revision as of 05:16, 27 October 2007 (view source) Silvapy (Talk | contribs) ← Older edit		Revision as of 05:21, 27 October 2007 (view source) Silvapy (Talk | contribs) (→'''Methods''') Newer edit →
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	Primers		Primers
-	crtN-F      3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’	+	3crtN-F      3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’
	crtN-R      5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’		crtN-R      5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
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-	PCR	+	'''PCR'''
	PCR of bch-? genes using - 20ng/ul pHM6		PCR of bch-? genes using - 20ng/ul pHM6
	1ul pHM6		1ul pHM6
Line 42:		Line 42:
	50ul total		50ul total
-	'''Dephosphorylation'''	+	'''Digestions'''
		+
		+	'''Dephosphorylation'''
		+
		+	50 ul pBR3222
		+	6 ul phosphatase buffer
		+	1 ul phosphatase
		+	3 ul H20
		+	Incubate 30" -1hr
		+	run gel
	== '''Results''' ==		== '''Results''' ==

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Revision as of 05:21, 27 October 2007

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 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

Dephosphorylation

  50 ul pBR3222
  6 ul phosphatase buffer
  1 ul phosphatase
  3 ul H20
  Incubate 30" -1hr 
  run gel

Results

Sequencing

Discussion