SBB09Ntbk-DaviddeRenzy

David De Renzy 14:34, 4 February 2009 (EST)

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David De Renzy 14:53, 18 February 2009 (EST)

• Reconstituted oligos to 100μM
• Ran basic PCR cloning reaction
• PCR Odd001F/Odd002R on E. Coli plasmid pAPEC-1 (AF218073) (1603 bp, EcoRI/BamHI
• Into PCR tube labeled M10040
  
  • Odd001F Forward EcoRI oligo for TshA ccataGAATTCatgAGATCTacacttttacctgtatacg
  • Odd002R Reverse BamHI oligo for TshA ctgatGGATCCtcagaatgaataacgaatattagcg

AND

• PCR Odd003F/BBa_G00101 on pBca1256-Bca1346 (3722 bp, EcoRI/BamHI)
• Into PCR tube labeled M10041
  • Odd003F Forward oligo for INP ccaaaGAATTCatgAGATCTtgtaATGaatctcgacaaggcg
  • BBa_G00101 Reverse sequencing of pSB1A* plasmids attaccgcctttgagtgagc

 
 Next step -> Run Analytical Gel

David De Renzy 15:38, 23 February 2009 (EST)

• Ran Analytical gel on M10040 & M10041
  • 5μL PCR product + 5μL blue loading buffer (concentration was weak)
• Both expected products were present
  • M10041 had a small fragment (~1000bp) of unknown DNA

• Performed Zymo clean-up on M10040 & M10041
  • Eluted final products into 33μL water

Digest and gel purify M10040 & M10041
Maybe, run another analytical gel & purify with remaining "zymo-cleaned" M10041
OR
Run a more stringent PCR for M10041 with higher annealing temperatures

David De Renzy 14:21, 25 February 2009 (EST)

• Digested M10040 & M10041
  • Used 8μL of each PCR product for digest
• Gel purified M10040 & M10041 Digests
• Also gel purified 15μL of M10040 PCR product (after zymo clean-up)