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I tried to make the probe several times but it failed again and again. It usually turns out that the probe after hydrolysis is very very short (maybe around 50nt). I did not check the RNA before hydrolysis except last time. However, I saw a band (though very weak) last time, but it still produce very very short probe after hydrolysis.I use Sp6 polymerase to synthesize RNA, and the original length of the DNA is 1.5kb. My expected length after hydrolysis is 500nt.

I'm guessing maybe it's the phenol contamination of the phenol:chloroform extraction step because my 260/280 value is low (around 1.6-1.7), and my 260/230 is also low, which is like 1.6-1.8. Also the peak is not at 260, it's almost at 270.

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I'm not all that familiar with in situ hybridization, but I'm missing a few details here. How did you synthesize the RNA probe (PCR, I assume?), and how long should it be? And that RNA is shorter than expected even before you use it in the in situ hybridization, do I understand that correctly? –  Mad Scientist Jul 16 '12 at 18:02
    
50bp does not sound that short for in situ... –  nico Jul 16 '12 at 18:33
    
I had large noise with such probe, but I can see the signal. However, I need to quantify the signal so I need better probe –  Pengyao Jul 16 '12 at 19:20
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2 Answers

Later I found out that the length of RNA read from the gel is not trustworthy. I guess it's because the RNA is in hybridyzation solution that even when it's running on the gel, it cannot reflect the actually length of the probe. The only way to test the probe quality is to do the in situ.

For the pheno contamination problem, I think it's a separate problem of myself. Once RNA can be synthesized in vitro, it means there should not be pheno contamination.

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I dont understand what you mean by hydrolysis in this context.

The way to go about is:

  • Clone the DNA, that will give rise to the probe, downstream of T7 (or any other) promoter
  • Linearize this plasmid by digesting at (or ahead of) the 3' of cloning site
  • Set up an In-vitro Transcription reaction with T7 polymerase (or whatever corresponds to the promoter you used)
  • Precipitate and purify the RNA (you can use DNAseI to get rid of the DNA).
  • To check the RNA size: mix ~2$\mu$l of RNA (less or more depending on your yield) with equal volume of 2x RNA loading dye (contains formamide 95%, EDTA, SDS, and the dyes).
  • Heat at 85⁰C for 5min. Let it cool and run in 1% TAE/TBE-agarose gel (no need to make MOPS-Formaldehyde gel)
  • dsDNA ladder wont be very accurate but may be used for size estimation (if linear, the mobility of dsDNA and ssDNA/RNA wont be very different. Formamide treatment keeps the RNA linear). Use ssDNA or RNA ladder for better accuracy.

Btw.. the absorbance ratios mentioned are okay.. you don't need an RNA of supreme quality for this application (in fact this suffices for most experiments)

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