DIFFERENTIAL DISPLAY

• 10U RNA guard (Pharmacia)
• 10U DNase I
• 10mM Tris-HCl buffer, pH 8.3
• 50mM KCl
• 1.5mM MgCl2
• 20ug Total RNA
• Add RNA water to 50ul
• 30^o C, 30Min.

   

• 10ul RNA Water
• 1ul DNA-free total RNA
• 4ul 5Xreverse transcription buffer
• 2ul 0.1M DTT
• 1ul 10mM dNTP stock
• 1ul Reverse transcriptase
• 1ul T12NA, T12NT, T12NG or T12NC (for separate reactions)
• 37^o C, 60min.

   

• 15ul ³³ P-dATP
• 50ul 10XPCR buffer
• 5ul 0.2mM dNTP
• 10ul Taq polymerase (5u/ul)
• 20ul H2 O

• 2ul RT PCR product
• 12ul PCR reaction stock&
• 45ul H2 O
• 3ul oligo-dTplus primers(T12NA, T12NT, T12NG or T12NC) in the repective tubes

• Divide the solution above in each tube to 3 small Eppendorf tubes,19ul each, add 1ul of each random primer (10mM stock) to each tube (in my case, three different random primers were used), and one drop of mineral oil to each tube.
• PCR cyclings

  • 94^o C, 5min., link to 40 cycles of:

     

  • 94^o C, 30sec.
  • 40^o C, 2min.
  • 72^o C, 30sec.

     

  • Link to 72^o C, 5min., hold at 4^o C

• Prepare 6% sequencing gel:

  • 30g urea
  • 6ml 5XTBE buffer
  • 9ml 38:2 acrylamide
  • 23ml water, mix gently
  • Add 120 ul 20% APS and 60ul TEMED

  • Add 5ul loading buffer (same as used in sequencing gel) to each PCR reaction tube.
  • Denature the reaction at 95^o C, 3min.
  • Prerun the gel at 1300V for 20min. in 1XTBE buffer.
  • Load the samples, paralell between corresponding samples.
  • Run the gel at 1300V for 4-5Hrs, 1100V in gradient gel.
  • Fix the gel with 10% ethanol for 15min.
  • Dry the gel in a gel-drier for 1hr.
  • Exposure to X-ray film overnight.
  • Cut the differential bands from the gel, put in 50ul H2 O, 37^o C, 1hrs, then boil for 5min.
  • Add 1/10 vol. of 3M NaAc, and 150ul 100% ethanol, -70^o C o/n.
  • Spin at 4^o C 5min.
  • Suck out the ethanol, spin dry. Then disolve the pellet in 10ul TE buffer.
  • Secondary PCR

    • 30ul Water
    • 1ul DNA fragment purified from gel
    • 2ul 2mM dNTP
    • 1ul oligo-T12NX primer (corresponding the primary reaction)
    • 1ul andom primer(corresponding the primary reaction
    • 5ul 5XPCR buffer
    • 10ul 50% glycerol
    • Add 1 drop of oil.

    This step can also be carried out by using a PCR stock solution to reduce the pepetting. To make the PCR stock, mix:

    • 840ul pure water
    • 54ul 2mM dNTP
    • 140ul 10XPCR buffer
    • 280ul 50% glycerol
    • For a 50ul reaction, use:

    • 46.5ul the stock
    • 1ul DNA fragment
    • 1ul 3'primer
    • 1ul 5' primer

     

    • PCR cycling, same as before(#13), 0.5ul Taq polymerase was added after hot-start
    • Run the samples with 1.5% agarose gel in 1/2TBE buffer

      Note: To have a pure PCR product, it will help to run the secondary PCR product together with the original in a sequencing gel and cut out the band again.

    • Northern blot and probe with ³² P-dATP labeled bands to confirm the difference.