Primer Extension

RNA isolation

Primer Labeling

  1. To 0.5pmole primer (0.5µl 10pmoles/µl) add 1µl 10x T4 polynucleotide kinase buffer from Roche kit.
  2. Add 2.5µl 32P-γ-labelled dATP (5µl if using 32P-γ-labelled dATP past it's first half life).
  3. Add DEPC treated water to make up to 9µl (4.5µl if using 10pmoles/µl primer).
  4. Add 1µl T4 polynucleotide kinase from same kit.
  5. Incubate for 30 minutes at 37°C.
  6. Remove top and bottom cap from NAP-5 column (Amersham-Pharmacia Biotech) to drain of storage liquid.
  7. Fill up with DEPC treated water 3 times and allow to drain.
  8. Add 500µl DEPC treated water to primer labeling reaction.
  9. Add primer labeling reaction to NAP-5 column and allow cold flow through to drain off.
  10. Add 1ml of DEPC treated water and collect eluate with purified hot primer.

    Reverse transcription with Superscript II from radioactive primer

  11. Take 30µg of RNA for highly expressed genes or 50µg for poorly expressed genes and adjust volume to 40 µl.
  12. Add 60µl of the eluate from the purification of labeled primer.
  13. Add 10µl (1/10 volume) of 3M sodium acetate.
  14. Add 250µl of 95% ethanol.
  15. Incubate overnight at -70°C.
  16. Spin for 30 minutes at full speed.
  17. Remove and discard supernatant carefully.
  18. Add 500µl 75% ethanol.
  19. Spin 10 minutes at full speed.
  20. Remove and discard supernatant carefully.
  21. Resuspend in 30µl of DEPC treated water.
  22. Incubate at 90°C for 3 minutes.
  23. Cool slowly to under 30°C.
  24. Add 18.5µl of DEPC treated water.
  25. Add 20µl of 5* Superscript II buffer.
  26. Add 10µl 0.1 M DTT.
  27. Add 20µl dNTP-mix for primer extension.
  28. Add 1µl of RNasin (RNase-inhibitor, Promega).
  29. Add 0.5µl Superscript II reverse transcriptase.
  30. Incubate at 42°C for 90 minutes.
  31. Add 1µl 0.5M EDTA and mix.
  32. Add 0.5µl RNaseA (Promega) and mix.
  33. Incubate at 42°C for 10 minutes.

    Purification by Phenol/Chloroform extraction

  34. Add 100µl Phenol to the reaction, mix by vigerous shaking.
  35. Spin 10 minutes at full speed to separate phases.
  36. Transfer the aquaous (top) phase to a new tube with 125µl 50:50 Phenol:Choloroform.
  37. Add 25µl dH2O to the tube containing the Phenol phase.
  38. Transfer the aquaous phase to the tube with Phenol:Chloroform, mix by vigerous shaking.
  39. Spin 10 minutes at full speed to separate phases.
  40. Transfer the aquaous (top) phase to a new tube with 150µl Choloroform.
  41. Add 25µl dH2O to the tube containing the Phenol:Cholorofom phase.
  42. Transfer the aquaous phase to the tube with Chloroform, mix by vigerous shaking.
  43. Spin 10 minutes at full speed to separate phases.
  44. Transfer the aquaous (top) phase to a new tube.
  45. Add 25µl dH2O to the tube containing the Chloroform phase.
  46. Transfer the aquaous phase to the tube with the aquaous phase from previously.
  47. Add 17.5µl (1/10th volume) 3M Sodium-Acetate and mix.
  48. Add 550µl (3 volumes) 95% ethanol.
  49. Leave at -70°C for 15 minutes or overnight.
  50. Spin 30minutes at full speed.
  51. Remove supernatant, and add 500µl 75% ethanol.
  52. Spin for 10minutes.
  53. Remove supernatant and airdry until no ethanol is left in tube.
  54. Dissolve in 6µl dH2O.
  55. Add 4µl stop solution from Amersham manual sequncing kit.

    Load and run on a manual sequencing gel along with a manual sequencing reaction preferably done on the relevant gene with the same primer according to manufactureres directions. Dry the gel and expose X-ray film to it for 1 to 2 days.

Solutions

Production and purification of labeled cDNA

dNTP-mix for primer extension
10µl 100mM dATP, 10µl 100mM dCTP, 10µl 100mM dGTP, 10µl 100mM dTTP, 10µl DEPC-treated water.

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