Home Protocols Size Selection and Gel Purification of Amplified cDNA Library (E6160)

Size Selection and Gel Purification of Amplified cDNA Library (E6160)

Protocol

  1. Mix 50 μl of amplified cDNA construct with 10 μl of Gel loading dye, blue (6X).
  2. Load 5 μl of Quick-Load pBR322 DNA-MspI Digest in one well on the 6% PAGE gel.
  3. Load two wells with 30 μl each of mixed amplified cDNA construct and loading dye on the 6% PAGE gel.
  4. Run the gel for 60 minutes at 120 V or until the front of the dye reach the bottom of the gel. Do not let the dye exit the gel.
  5. Remove the gel from the apparatus and stain the gel with SYBR® Gold nucleic acid gel stain in a clean container for 2–3 minutes and view the gel on a UV transiluminator.
  6. Cut the bands corresponding to ~110–119 bp. The 110 and 119 nucleotide bands correspond to adaptor-ligated constructs derived from the 21 and 30 nucleotide RNA fragments, respectively. Do not cut the 89 bp band out, as this is adaptor dimer. (Figure 1 on product page).
  7. Place the gel slice in a 1.5 ml tube and crush the gel slice with the RNase-free Disposable Pellet Pestles and soak in 250 μl DNA Gel Elution Buffer (1X).
  8. Rotate for 2–18 hours at room temperature.
  9. Transfer the eluate and the gel debris to the top of a gel filtration column.
  10. Centrifuge the filter for 2 minutes at 14.000 rpm.
  11. Recover eluate and add 1 μl Linear Acrylamide, 25 μl 3M sodium acetate pH 5.2 and 750 μl of 100% ethanol.
  12. Vortex well.
  13. Precipitate in a dry ice/methanol bath for at least 30 minutes.
  14. Spin in a microcentrifuge (>14.000 x g) for 30 minutes at 4°C.
  15. Remove the supernatant taking care not to disturb/remove the pellet.
  16. Wash the pellet with 80% ethanol by vortexing vigorously.
  17. Spin in a microcentrifuge (>14.000 x g) for 30 minutes at 4°C.
  18. Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.
  19. Resuspend pellet in 10 μl TE Buffer. Perform the following quality control analysis on your sample library to quantify the DNA concentration.
  20. Load 1 μl of the resuspended construct on an Agilent Technologies 2100 Bioanalyzer using a DNA specific chip such as the Agilent DNA-1000 (Figure 2 on product page).
  21. Check the size, purity and concentration of the sample. The final product should be a distinct band. The final product should be a distinct band. If you see undesirable peaks (bigger or smaller than your expected range sizes) perform a second round of size selection.