Dioscorea alata lirm.sp tissue RNA extraction method

Dioscorea alata lirm.sp tissue RNA extraction method

  • CN 104,164,419 A
  • Filed: 07/23/2014
  • Published: 11/26/2014
  • Est. Priority Date: 07/23/2014
  • Status: Active Application
First Claim
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1. purple sole potato is organized the extracting method of RNA, it is characterized in that comprising the steps:

  • (1) RNA extracting solution 65 is spent to water-bath 15 min;

    Described RNA extracting solution is 2% SDS, 100 mM Tris, 25mM EDTA, 2M NaCl solution;

    (2) often get the purple sole potato of 0.5 g organization material and put into the mortar that has added in advance 0.03 g polyvinylpyrrolidone (PVPP), add liquid nitrogen and be ground to rapidly powder;

    (3) powder transfer, to centrifuge tube, adds 0.6 ml RNA extracting solution and 24 μ

    l beta-mercaptoethanols, water-bath 10 min after the vortex several seconds;

    (4) add after 0.6 ml volume ratio 24;

    1 chloroform isoamyl alcohol after concuss 1 min centrifugal 8 min of 13000 rpm;

    (5) get supernatant, add 3 mol/L pH5.2 KAC of ethanol and 0.1 times of volume of 2.5 times of volumes, mix ice bath 10 min, centrifugal 10 min of 13000 rpm;

    (6) abandon supernatant, add 1 ml 70% washing with alcohol, centrifugal 5 min of 13000 rpm, abandon supernatant, more slightly centrifugal, make raffinate combine in the pipe end, suck with pipettor, are placed on Bechtop with aseptic wind 2-5 min, add 43 μ

    l water dissolution;

    (7) add 5 μ

    l DNase Buffer, 2 μ

    l DNase, gentleness mixes, 37 DEG C of 30 min;

    (8) add 350 μ

    l water, 200 μ

    l acid phenol, 200 μ

    l chloroform isoamyl alcohols, concussion 1 min, centrifugal 10 min of 13000 rpm;

    (9) get supernatant, add isopyknic chloroform isoamyl alcohol, chloroform isoamyl alcohol volume ratio 24;

    1, concussion 1 min, centrifugal 10 min of 13000 rpm;

    (10) get supernatant, add 3 mol/L pH5.2 KAC of ethanol and 0.1 times of volume of 2.5 times of volumes, mix ice bath 10 min, centrifugal 10 min of 13000 rpm;

    (11) abandon supernatant, add 1 ml 70% washing with alcohol, centrifugal 5 min of 13000 rpm, abandon supernatant, make raffinate combine in the pipe end, suck with pipettor, are placed on Bechtop with aseptic wind 2-5 min, add suitable quantity of water dissolving.

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