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Porblems making DIG probe - please help

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Porblems making DIG probe - please help

Postby chaka8 » Tue Jul 29, 2008 2:02 am

Hi,
I am having problems with making DIG RNA probes (Roche). I get no bands when running a gel after purification other than the weak band of the linearised plasmid.
I am using T7 and SP6 polymerase. I seem to have narrowed down the problem to the process of making the probe (I have no band before purification either).
So it’s the DIG mix, the polymerase, the buffer or the process.
Today I verified the incubator temp and it was 42c instead of 37c, would incubating for two hours at 42c be the problem? Do T7/SP6 polymerases have no activity at 42c??
And if this is not the problem, what else could it be?
I will be trying a new DIG kit and new polymerases.
Thanks!!!
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Postby snowcapk » Tue Jul 29, 2008 7:47 am

A couple of questions:
  • Have you tried the controls that come with the kit? If you didn't use the controls, are you sure that you've matched the correct polymerase to the correct restriction digest product?
  • Are you sure your DIG "mix" contains the other NTPs? Are you sure you have the DIG mix for RNA and not the DIG mix for DNA? Our lab has both mixes floating around, maybe yours does too...and I bet Sp6 wouldn't like dNTPs one bit.
  • How's the buffer? Ours (possibly from the mMessage mMachine kit) forms a flocculate whenever it freezes that is difficult to resuspend - it has to be heated and vortexed. The reason I ask is that someone in our lab was spinning it down (!) and drawing off the supernatant to use in her reactions, ruining the whole aliquot. When it comes to retardation, the perpetrator is never the only victim ;) Ask other lab members what they do to find out if they're sabotaging you.
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Re: Porblems making DIG probe - please help

Postby chaka8 » Tue Jul 29, 2008 11:42 am

Thanks snowcapk,

Haven’t tried the control, I don't think that the Roche kit comes with one but I'll check.
I'm sure about matching the polymerase to the liberalized vector.
The DIG mix contains everything and it’s the RNA mix.
I know that polymerase buffers have a precipitant and I vortex and even heat a bit before use.

Do you have any insight regarding incubation at 42 instead of 37?
How is it best to purify the vector after restriction? I'm using a cleanup kit and eluting with DEPC water...?

It does suck when someone else messes you up!
Thanks again for the suggestions.
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Postby snowcapk » Wed Jul 30, 2008 8:06 am

Hi chaka8,

It sounds like you've considered everything that I would have - sorry I can't be more helpful! I don't actually know about the incubation temp., but it doesn't seem like it should be a problem. Your cleanup method sounds fine - I use a column from a Qiagen PCR purification kit or Zymo Research DNA Clean-up and Concentrator kit personally, but phenol chloroform extraction followed by isopropanol precipitation is another option. I also elute with DEPC water.

If you could find a positive control, even a linearized plasmid someone else in your lab had used successfully, I think it'd be worth repeating the IVT reaction. We use the Roche DIG labeling mix with the Ambion mMessage mMachine IVT kits so I'm not familiar enough with your kit to know what might be wrong.

How many times have you attempted the IVT?
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Re: Porblems making DIG probe - please help

Postby chaka8 » Thu Jul 31, 2008 3:43 am

OK, did the whole thing over again, everything new.
I ran a non-denaturing ethidium bromide gel.
In one lane I ran just some of the DIG RNA mix and I see nothing, shouldn’t I see a strong band of the free RNA neucleotides??
In the other lanes I ran 1ul of the reactions (after 2.5 hours at 37c), I do see weak smears, this would mean that I have introduced nucleases into the reaction and that the RNA probe is being cleaved into pieces during the incubation, correct??
If so, I have no idea where it is coming from, everything is new, any thoughts on how to avoid this?
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Postby snowcapk » Sat Aug 02, 2008 1:59 am

Ethidium intercalates into nucleic acids; I don't think it binds to free nucleotides well. This is probably why you cannot see them in an EtBr gel. If you really think that you might not have any nucleotides in there, you could use a spectrophotometer to check the concentration.

You need to run a special gel for mRNA. It needs to be a denaturing (formaldehyde) gel and it needs to be performed in a gel box that is RNase-free, with RNase-free buffer, etc. Before being loaded onto the gel, your sample needs to be heated in formaldehyde to denature it. If you don't run the gel this way, you'll see smears no matter what. The apparent fragment length will also be inaccurate due to secondary structures affecting migration rate. Did you check the concentration of mRNA in your sample by spectrophotometer? If you aren't getting enough product, you can always run the reaction for a longer period of time.
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