3 replies to this topic

[biznatch]

I'm using Klenow to label a ~3kb PCR product to make DIG labelled probes for DNA FISH.  I got a protocol from a lab mate who's done it successfully, who got the protocol from someone in another lab, who got the protocol from another lab...you know how it is...and some of the steps don't make sense to me.

We start with the BioPrime DNA Labeling System from Invitrogen to do the actual labeling (but make our own dNTP mix with DIG UTP) then purify the probe (remove unincorporated nucleotides) using illustra ProbeQuant G-50 Micro Columns from GE.  I would think at this point it'd be all finished and I'd have my probe in TE, but the protocol includes additional steps, can anyone provide insight as to what these steps are for?  After column purification:

1. Add EtOH, yeast tRNA, NaOAc
2. Spin at 4'C, remove supernatant.
3. Resuspend pellet in water, NaOAc, EtOH.
4. Add more EtOH, salmon sperm DNA, COT1 DNA, yeast tRNA, NaOAc.
5. Spin at 4'C, remove supernatant.
6. Wash in 70% then 100% EtOH, dry pellet.
7. Resuspend in formamide.
8. Denature at 85'C, 10 min, then 2 min on ice.
9. Add 30 uL FISH hybridization mix, incubate 37'C 30 min.
10. Store at -20.

I have a few ideas.

The protocol for the BioPrime kit (linked above) says to purify either using EtOH/NaOAc washes OR to use a column, but this protocol uses both methods, and adds yeast tRNA (as a carrier?) to the first wash.  Is there any reason to use both purification methods?

The protocol uses salmon sperm and COT1 DNA, these are often use to block non-specific binding in FISH but would be added during hybridization I think, why are they being used here?

The last few steps (resuspend in formamide, denature, add to FISH mix) are usually part of the actual FISH protocol, not making the probe.  When doing FISH, I was told to use some of the probe as made above, which is in formamide and FISH mix, and add that to more FISH mix.

It seems to me that as the protocol has been passed down people are just adding steps on and mixing things up without bothering to wonder why they're doing any particular step, so when I ask my lab mate I get, "I don't know, it just works", which is fine if it works, but it's extra work, and I'd like to know what all these steps are doing rather than mindlessly following instructions.

As a bonus, feel free to post any other "hand-me-down" protocol horror stories  

[bob1]

You are correct about the proceedure being a further purification of the DNA using tRNA as a carrier.  Steps 4,5,6 appear to be another version of the same purification with the DNA/RNA being carriers again.

Steps 8, 9,10 could be done on the day you are doing FISH I guess, but it may be too concentrated in the form described above and needs to be diluted forther before use, so that this is cutting out some steps you will have to do anyway at a later point.

I would check this protocol against ones from other sources such as the Roche DIG manuals, which are pretty good.

[biznatch]

Ok thanks I will see what other protocols I can check it against.

For the temperature parts of of steps 8 and 9, in particular the denaturing, would storing it at -20 make this pointless?  Is it going to stay denatured?  Their FISH protocol calls for heating the probe + mix at 65'C for 10 minutes then 37'C for 30 minutes before hybridizing, does this make the 85'C + ice, etc. redundant? Or does the heating at 85'C and at 65'C serve different purposes?

[bob1]

There will be some hybridisation between probes if you allow them to cool slowly or sit at a low temperature for a while, freezing should (in theory) prevent this.  You can always re-denature if you are worried.