Team:Heidelberg/Templates/DelH overview11

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Gibson Strategy

  • PCR amplification of entire 18 Kb DelH fragment G0, as well as sub fragments G1a and G1b
  • PCR amplification of backbone pSB6A1-lacI-mRFP, simultaneously inserting Gibson
  • Gibson assembly of construct DelH + backbone to DelH plasmid
  • Electroporation of E. coli TOP10 with DelH plasmid
  • Characterization of positive (red) colonies by colony PCR


Vector Map and Primers

Vector map of the pHM02:DelH-pSB6A1-AraC-lacZ Gibson-plasmid.
Identifier Order Date Note Sequence
HM02_DelH_Gib1.1_rev 09-07-2013 Gibson-Primer DelH TGCTGCGCCTGCATACGGCCAAACA
HM03_DelH_Gib1.2_fw09-07-2013 Gibson-Primer DelH AGCGGCAGGGACGACGTGGT
HM04_DelH_Gib1.2_rev09-07-2013 Gibson-Primer DelH CATAGAGGTTGTAGAGA
HM05_DelH_Gib2.1_fw09-07-2013 Gibson-Primer DelH AGAACGCCGTCTTCAGGCTCCTG
HM06_DelH_Gib2.1_rev09-07-2013 Gibson-Primer DelH CAATGCTTTG CCGCTCGAA
HM07_DelH_Gib2.2_fw09-07-2013 Gibson-Primer DelH TCGCCACGGCAGCTGTTCGA
HM08_DelH_Gib2_end_rev09-07-2013 Gibson-Primer DelH TCAGTCCAGCGCGTACTCCAG
HM09_AraC_RBS_DelH_rev09-07-2013Gibson-Primer rev, introduces a
new RBS and has the AraC promotor
and the beginning of DelH		 TTGCAAAGCGCTCGGCGATTTGGCGCAGGCGGCCACGGTCCAT
ttaactTTCTCCTCTTTAATactttgagctagcccaaAaaaacggtatggagaa
acagtagagagtt
HM10_RBS_lacZ09-07-2013 Gibson-Primer fw for the pSB6A1
Backbone with the end of DelH,
RBS(1) and the beginning of lacZ 		TGGAGTACGCGCTGGACTGA TCTAGAG AAAGAGGAGAAA TACTAG ATGACCATGATTA


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