Reiter:32P

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(New page: #5’ 32P-label pre-tRNA substrates (2 substrates): (i) Dephosphorylation: *0.5 µl 40 µM RNA *27.5 µl water *0.75 µl 2M trisHCl, pH 8.1 *1 µl CIP (calf intestine alkaline phosphatase)...)
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Revision as of 17:35, 22 March 2012

  1. 5’ 32P-label pre-tRNA substrates (2 substrates):

(i) Dephosphorylation:

  • 0.5 µl 40 µM RNA
  • 27.5 µl water
  • 0.75 µl 2M trisHCl, pH 8.1
  • 1 µl CIP (calf intestine alkaline phosphatase)
  • 37 ºC for 30 min.

+20 µl 50 mM KOAc/200 mM KCl, pH 7 (C+S buffer) “Gentle” Phenol/CHCl3 extraction, followed by ethanol precipitation.

  1. Kinase reaction (This is the HOT HOT step):
  • 2 µl 10x kinase buffer (provided by USB)
  • 15.5 µl water
  • 2 µl γ-32P-ATP (25 µM)
  • 0.5 µl T4 kinase (USB)
  • Split into 2x10µl, add to pellet from (i).
  • 37 ºC for 30 min.

Purify on P6 spin columns (Bio-Rad) Perform this spin column procedure 4-6 times to ensure NO free 32P-ATP is present. Aliquot and Ethanol precipitate, spin down, 80% EtOH wash (x2) Carefully pour off all Ethanol, air dry O/N in hood FULLY protected. Store at -20ºC (should be good for two weeks). Dissolve the aliquot in 40-60 µl water immediately prior to use.

I. Cleavage reaction (e.g. for 50 nM [Enzyme] final). Final buffer condition: 1x THE, 10 mM MgCl2, 0.1 M NH4OAc.

  1. Holoenzyme folding (make 1 µM first, then dilute):
  • 1.25 µl 10x THE buffer
  • 1.25 µl 10 µM P RNA
  • 1.25 µl 10 µM P protein
  • 6.25 µl water

85-90 ºC for 2 min. Room temperature for 3 min. + 1.25 µl 100 mM MgCl2 50 ºC for 10 min. + 1.25 µl 1 M NH4OAc 37 ºC for 5 min. 1.25 µl + 11.25 1xTHE/MgCl2/NH4OAc

  1. Substrate folding:

2.5 µl 10x THE buffer 4 µl 5’ 32P-labeled substrate 13.5 µl water 85-90 ºC for 2 min. Room temperature for 3 min. + 2.5 µl 100 mM MgCl2 + 2.5 µl 1 M NH4OAc 37 ºC for 5 min. Save 1 µl + 5 µl 9M urea/50 mM EDTA/BPB/XC, this sample is t = 0.

(iii) Reaction: Start reaction by mixing 5 µl Enzyme + 5 µl substrate. Incubate at 37 ºC. At 0.25, 1, 4, 16 min, mix 2 µl reaction mixture + 5 µl 9M urea/50 mM EDTA/BPB/XC.

(iv) Gel and TLC: For +9 and +1-tRNA substrates, separate substrate from product on 15% denaturing gel, run BPB about halfway done (for +1-tRNA substrate analysis, see image below). For +1-tRNA substrate, spot 1.5 µl mixture on cellulose PEI plate, run TLC in 5% acetic acid/100 mM

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