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Edinburgh-Genome-Foundry / OT2Metclo

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These are the parts of the Metclo Assembly, devided into possible automated and non automated sections

DanyMatute opened this issue · comments

commented

PROTOCOL
in Zotero library

Bold steps could potentially be done in the OT2
*are notes on the steps
the steps are followed by sub-steps which could/could not be automated

  1. Get inserts
  2. Prepare chemical competent DH10B-MOsp807II
  3. Prepare MetClo Assembly vectors within the chemically competent DH10B-MOsp807II
    miniprep vectors from picks
    chemical transformation vector in to cells
    *must have different antibiotic selection market than insert plasmids
  4. purify insert and metclo assembly vector plasmids
  5. Conduct metclo assembly if insert and metclo assembly vector THIS IS MY DISSERTATION
    *the protocol changes slightly for the size of the assembly
  6. Purify and Analise Assembled DNA (insert in metclo assembly vector)
    *if analysis is good move to step 7
  7. Prepare a final assembly vector (with chemically compitent DH10B-MOsp807II) that will be accepting the plasmids from 5
    *with a different antibiotic selection market to step 5 plasmids
    *preparation as done in step 3
  8. purify step 5 plasmids (another purification method different from step 6)
    *the purification kit is different for p15a or F replication origin plasmids
  9. repeat DNA assembly by metClo (step 5) with plasmids in step 7 and step 8