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Revision as of 05:27, 27 October 2007 (view source) Silvapy (Talk | contribs) ← Older edit		Revision as of 05:47, 27 October 2007 (view source) Silvapy (Talk | contribs) (→'''Methods''') Newer edit →
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	Primers		Primers
		+	crtN-F    5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
		+	crtN-R    3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’
-	3crtN-F     3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’	+	bchB-F     5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA
		+	bchB-R    3'ttaGGATCCccgttcgccttggtttgacttact5'
		+
		+	bchE-F    ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca
		+	bchE-R    gctagCTCGAGtattttcatcatgcctctcgt
		+
		+	bchI-F    ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT
		+	bchI-R    ttaGGATCCtcggggctgagaaggcgggagca
-	crtN-R     5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’	+	bchL-F    ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA
-		+	bchL-R     ttaGGATCCttgggcagaaggtgtggaagca
-	bch B      3'ttaGGATCCccgttcgccttggtttgacttact5'	+
		+	bchM-F    ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC
		+	bchM-R    ttaaGGATCCtctcttcggcttaatttccaacag
		+	bchN-F    accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac
		+	bchN-R    attaggatccTCATTCCAGCCACCCCGCTT
	'''PCR'''		'''PCR'''
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	Ligations (3hrs @ R.T)		Ligations (3hrs @ R.T)
-
	== '''Results''' ==		== '''Results''' ==

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Revision as of 05:47, 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

crtN-F     5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
crtN-R     3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’

bchB-F     5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA
bchB-R     3'ttaGGATCCccgttcgccttggtttgacttact5'

bchE-F     ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca 
bchE-R     gctagCTCGAGtattttcatcatgcctctcgt

bchI-F     ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT
bchI-R     ttaGGATCCtcggggctgagaaggcgggagca

bchL-F     ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA   
bchL-R     ttaGGATCCttgggcagaaggtgtggaagca

bchM-F     ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC
bchM-R     ttaaGGATCCtctcttcggcttaatttccaacag

bchN-F     accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac
bchN-R     attaggatccTCATTCCAGCCACCCCGCTT

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