Preparation of Fluorescent DNA Probe from HUMAN mRNA or Total RNA using Direct I

Preparation of Fluorescent DNA Probe from HUMAN mRNA or Total RNA using Direct Incorporation (Max Diehn/Ash Alizadeh protocol; 3/15/01)

I. Preparing fluoresenctly labeled cDNA (probe):

1. To anneal primer, mix 2ug of mRNA or 50-100mg total RNA with 4ug of a regular or anchored Oligo-dT primer in a total volume of 15.4 ul:

   Cy3

   Cy5

   mRNA (1

   x

   y l

   (2 mg of each if mRNA, 50-100mg if total RNA)

   Oligo-dT (4

   1

   1

   (Anchored: 5'-TTT TTT TTT TTT TTT TTT TTV N-3')

   ddH₂O (DEPC)

   to 15.4

   to 15.4

   Total volume:

   15.4

   15.4

   ^oC for 10 min and cool on ice.

   Reaction mixture

   l

   . . . . .

   Unlabeled dNTPs

   Vol.

   Final conc.

   5X first-strand buffer*

   6.0

   dATP (100 mM)

   25 uL

   25 mM

   0.1M DTT

   3.0

   dCTP (100 mM)

   25 uL

   25 mM

   Unlabeled dNTPs

   0.6

   dGTP (100 mM)

   25 uL

   25 mM

   Cy3 or Cy5 (1 mM, Amersham)

   3.0

   dTTP (100 mM)

   10 uL

   10 mM

   Superscript II (200 U/uL, Gibco BRL)

   2.0

   ddH2O

   15 uL

   Total volume:

   14.6

   Total volume:

   100 uL

   * 5X first-strand buffer: 250 mM Tris-HCL (pH 8.3), 375mM KCl, 15mM MgCl2)

   ^oC for 1 hr. l SSII (RT booster) to each sample. Incubate for an additional 0.5-1 hrs. ml of 0.1N NaOH, 2mM EDTA and incubate at 65-70^oC for 10 min. If starting with total RNA, degrade for 30 min instead of 10 min. ml of 0.1N HCl. l l l lll l g/l)