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发布时间:2019-01-20 12:00:00来源:深圳市第二人民医院 分享:

  Methods 

  1、Ligation Independent Cloning (LIC) Procedure: 

  Part I:  

  

  1. design primers of the expression vector and the target sequence.
  2. PCR amplification to get the target sequence and the expression vector.
  3. Gel purify the PCR produces using standard methods.

  Part II: T4 DNA Polymerase Treatment of vector and PCR Inserts: 

  

  1. Use about 0.037 pmoles of vector per 20μl reaction.
  2. Use 0.12-0.48 pmoles of gel purified PCR-insert per 20 μl reaction. Gel purification of PCR-products is necessary to decrease the background of non-specific products.

  Make Vector and Insert Mixes: 

  


  a.Vector Mix:       1X 

  T4 Buffer             2 μl 

  DTT (100mM)       1 μl 

  T4 Polymerase      0.4 μl 

  dGTP             2 μl 

  ddH2O     fill to make 20ul 

  b.Insert Mix :           1X 

  T4 Buffer               2 μl 

  DTT (100mM)           1 μl 

  T4 Polymerase         0.4 μl 

  dCTP                  2 μl 

  ddH2O       fill to make 20 μl 

  


  Part III: Annealing vector to insert

  1. Spin down samples.
  2. Mix 5μl vector with 5μl insert.
  3. Incubate at 22°C for 5 min.
  4. Add 2.5μl EDTA (25 mM) and mix well.
  5. Incubate at 22°C for 5 min.