IGEM:Indian Institue of Technology Madras/2009/Notebook/PLASMID - Plasmid Locking Assembly for Sustaining Multiple Insert DNA/2009/09/29

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  • PCR done of the following parts (to be used for further digests/ligations etc)
  • S03879,K145151,I13507,I13502,I13504

Program used: IGEM1 (edited: elongation time = 45sec) /mistake: the program was run for 5 cycles with elongation time = 1min, then program running was stopped and running was started again with elongation time= 45sec)

  • Transformed K084014 in 1A2 and K084012 in 1AK3
  • Restriction digested S03879 (amplified part from PCR) at S, PSB1A3 digested at S, I13507 (PCR amplified) digested at X, PSB1A3 digested at X
  • PCR purified S03879 digested at S and I13507 at X to remove smaller parts and concentrated to 25 micro litre.
  • Ligated

S03879 (cut at S)+ PSB1A3 (cut at S); I13507 (cut at X) + PSB1A3 (cut at X); S03879 (cut at S) + I13507 (cut at X)

  • Ran gel; order of loading the gel:

ladder-(empty)-PSB1A3-PSB1A3 (cut at X)-S03879 (cut at S)-I13507 (cut at X)- S03879 (cut at S)+ PSB1A3 (cut at S) - S03879 (cut at S) + I13507 (cut at X)- I13507 (cut at X) + PSB1A3 (cut at X) ///Doubt : 1A3 or 1A2????/// - Ladder