Home Protocols Construction of Illumina Library or Expression Library from 1-5 μg genomic DNA (E6000)

Construction of Illumina Library or Expression Library from 1-5 μg genomic DNA (E6000)

Overview

DNA fragmentation: Expression Libraries can be generated from any fragment insert size. Common methods to shear DNA include nebulization, acoustic shearing, and hydroshearing. Please refer to specific protocols for DNA fragmentation and purification.

Protocol

  1. DNA fragmentation. Expression Libraries can be generated from any fragment insert size. Common methods to shear DNA include nebulization, acoustic shearing, and hydroshearing. Please refer to specific protocols for DNA fragmentation and purification.
  2. End Blunting reaction following DNA fragmentation
    1. For each 100 µl reaction, assemble the following components:
      30 µl Fragmented DNA sample
      10 µl Phosphorylation Buffer
      4 µl Deoxynucleotide Solution Mix
      5 µl T4 DNA polymerase
      1 µl DNA polymerase I, Large (Klenow) Fragment
      5 µl T4 polynucleotide Kinase
      45 µl dH20
    2. Mix gently and incubate for 30 minutes at 20°C.
    3. Purify DNA by phenol/chloroform and ethanol precipitation or DNA purification column. Resuspend DNA in 32 µl TE buffer.
  3. dA Tailing adds a single dA to the 3' end of the blunted, phosphorylated DNA using Klenow Fragment (3'-> 5' exo-).
    1. For each 50 μl reaction, assemble the following components:
      32 µl blunted, phosphorylated DNA
      5 µl NEBuffer 2 for Klenow exo-
      10 µl μl dATP Solution
      3 µl Klenow Fragment (3´→ 5´ exo-)
    2. Mix gently and incubate for 30 minutes at 37°C.
    3. Purify DNA by phenol/chloroform and ethanol precipitation or DNA purification column. Resuspend DNA in 32 µl TE buffer.
  4. Ligate dA tailed- or blunt ended DNA into a linearized expression vector with either a 3' dT overhang such as described in Jeung et al. for TA cloning or blunt ends:
    1. For each 50 µl reaction, assemble the following components:
      10 µl dA tailed or blunt DNA
      x µl linearized expression vector
      25 µl Quick Ligation Reaction Buffer
      5 µl Quick T4 DNA Ligase 
      x µl dH20
    2. Mix gently and incubate for 15 minutes at 20°C.
    3. Purify DNA by phenol/chloroform and ethanol precipitation or DNA purification column. Resuspend DNA in 25 µl TE buffer.
  5. Transformation:
    Transform competent E. coli cells such as T7 Express with 5 µl of ligation as follows:
    1. Thaw a tube of competent E. coli cells on ice for 10 minutes.
    2. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture.
    3. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
    4. Place the mixture on ice for 30 minutes. Do not mix.
    5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
    6. Place on ice for 5 minutes. Do not mix.
    7. Pipette 950 µl of room temperature SOC into the mixture.
    8. Place at 30°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    9. Warm selection plates to 30°C. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 30°C. Alternatively, incubate at 25°C for 48 hours.
    References:
    1. Clark, J. M., et al. (1987) J. Mol. Biol. 198, 123-7.
    2. Jeung, J. U., et al. (2002) Plasmid 48, 160-3.
    3. Sambrook, J. and D. Russell (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, CSHL Press.