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Home > Services > Whole Genome Service > BAC library construction
 
HMW DNA sources
   Library source
  - Plant - Young Leaf
     - Plug preparation : Nucleic DNA Extraction
  - Mammalian - Sperm Cell,
   Blood
     - Insert Preparation : Partial digestion
  - Insect - Egg Cell
     - Insert size : 100~200kb
  - Bacteria
     - Vector : Low Copy number Vector (pIndigoBAC536)
  - Virus
     - Library format : Cell or DNA
         
      Time required
        - 8 weeks will be required once QC report is approved.
 
 
Step 1 : Plug Preparation
  a.
b.

Plug preparation :Separates HMW (High Molecular Weight) DNA from the tissue.
Plug quality check : Complete digestion with restriction enzymes (BamHI, EcoRI, HindIII)

   
Step 2 : Insert Preparation 1
Partial digestion : Pilot partial digestion with serial dilution of restriction enzymes
(BamHI, EcoRI, HindIII)
       
Step 3 : Insert Preparation 2
  a.

b.

Mass partial digestion: Select the restriction enzyme dilution condition that produce
the most DNA fragments in the range of 100-400kb in the pilot partial digestion.
Size selection : Recover two gel fraction; A traction (150~250kb) and B traction (250~350kb)

   
Step 4 : Ligation & transformation
  a.
b.

Eluted insert DNA ligate to BAC vector (pIndgo BAC536)
Transformation to DH10B T1 resistant competent cell

   
Step 5 : Insert Size Check
  Pick the white colonies and digest the BAC plasmid DNA by NotI restriction enzyme.
 
Library report


Insert size (Average insert size 100kb <)
  Insert ratio (95%>)
   
Provision of library
  Provision of cell stock or DNA