[northern] Northern Hybridization for tRNA

[Seung Jae Jeong]

Northern Hybridization for tRNA

 

                                                           Sep. 24. 2001.

Joong-Won Lee

 

 

 

*Sample preparation

 

-Total RNA preparation

 

   a.RNA prep. by Trizol method(1)

   b.The pellet is dissolved in appropreate volume of 10mM NaOAc(pH4.5) 1mM EDTA or 250mM Tris-Cl(pH9.6). store at -70C⁰

   c.Control sample:deacylation:250mM Tris-Cl(pH9.6) incubation at 37C⁰ for 30min

 

 

*Gel preparation

 

-10% polyacrylamide(19:1), 8M Urea, 100mM NaOAc(pH5.0), 1X TBE.(2,3)

 

   a.12.5ml 40% polyacrylamide(19:1) / 24g Urea/ 1.66ml 3M NaOAc(pH5.0)/ 5ml 10XTBE

   b.Adjust the volume to 50ml with H₂O.

   c.add 400ul 10% APS and 30ul TEMED

 

 

*Electrophoresis

 

-Running Buffer:1X TBE.

 Sample buffer:0.1M NaOAc(pH5.0)/ 8M Urea/ 0.05% XC,BPB(4)

   a.Sample buffer:RNA sample=1.5:1

   b.running for 5hr at 500V. 

 

 

*Transfer

 

-Electronic transfer/transfer buffer:40mM Tris-acetate pH8.1

   a.Semi-dry transblotter 3hr (width X length = mA)

 

 

*Labeling of antisense oligonucleotides

 

-Probe Labeling:gamma32P end labeling:incubation at 37C⁰ for 30min

a.phosphorylation reaction

oligonucleotide 100ng(2ul)/

gamma-32P ATP(3ul)/

nuclease-free water(3ul)/

T4 polynucleotide kinase(10unit/ul)1ul/

Incubation at 37C⁰ for 30min

stop the reaction by adding 1ul of 0.5M EDTA

b.removal of unincoporated label

   sephadex G-25 column

 

 

*UV crosslinking and Hybridization

 

-UV crosslinking the transferred nylon membrane

-Hybridization sol:30% formamide/ 5Xdenhardt′s sol/ 5X SSC/ 0.1% SDS

   a.prehybridization:add 100ul ssDNA(10mg/ml stock) to 10ml hybridization sol and incubation at 37C⁰ for 2hr

   b.hybridization:add probe and incubation at 37C⁰ for 17hr

c.washing and expose:

2X SSC(0.1% SDS)/ 1X SSC(0.1% SDS)/ 0.1X SSC(0.1% SDS) 

 

Reference

 

1.      Anal.Biochem. 1987.    161:156-158

2.      P.N.A.S. 1987.         84:2185-1051

3.      J.B.C. 1991.           266:24712-24718

4.      M.C.B. 1996          16:907-913

 

 

 

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