HELP needed!

I've been using the ROCHE DIG High Prime DNA labeling and Detection Starter Kit I but unfortunately i keep getting light purple membranes with no bands. I don't think there's a problem with the Southern blot because all the PCR products were transferred and in fact there only ONCE when i did get bands but the rest of the time afterwards only gave me purple membranes though i had used the same probes and PCR products as before. Any suggestions of what I did wrong or what i didnt do right?

Thanks in advance!

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Hello Lene,

maybe you can describe what you exactly did?

The "usual" reasons for a high background can be:

- Inefficient hybridization: You should try another hybridization temperature and be sure not to let the membrane dry between prehybridization and hybridization.
- Wrong type of nylon membrane: Some types of nylon membrane may cause high background.
- Inefficient blocking: Block for a longer time and prolong also the washing steps.
- Ineffective stringency washes: Check temperature of stringency washes, always prewarm wash solution.

When using laboratory trays for the detection, they should be very clean. Anti-DIG-AP binding and color development should always be done in separate trays.

I hope that those are helpful suggestions to you...?

Greetings,
Chakchel

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Dear Chakchel,

Thanks for your reply. The hybridization temperature that i tried should be fine because I had been using the same Hyb temp for many trials i did and there was once that showed results so I think that should mean that the Hyb temp is ok and maybe something else is wrong in my technique?

What type of nylon membrane would you suggest for DNA-probe hybridization for the ROCHE DIG kit?

I will try out a longer blocking time, hopefully it would show less background. One of my friends had suggested that possibly the DNA had not been fixed properly to the membrane. She suggested I use the short wave UV at 5 minutes compared to previously where I had used the transilluminator function at 2 minutes.

I was wondering whether the DIG Easy Hyb Solution is reusable as it is not stated on the manual... Any experience using reused reagents from the Kit>?

Thank you again.
Best regards,
Charlene.

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Another brief question:
How do you prepare the reused DIG Easy Hyb containing DIG-labelled probe? From the protocol, it says to denature at 68C for 10mins before use. Does this mean that we do not need to chill on ice first and just immediately pour the warm solution onto the membrane?

Charlene.

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Hi Lene,

I hope you still have some time to let me look it up... I have to confess that's some time ago that I did this regularly. So I will have look in my old lab books... then I hopefully will be able to tell you the membrane type for example. I also can't remember that I ever had reused the DIG Easy Hyb Solution... hmmm
I will answer as soon as I find out (or maybe someone else is faster ).

Greetings,
Chakchel

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It's me once again

The membrane I used was Amersham Hybond N+.

And I always used fresh prepared solutions. The probe I denatured for 10min at 95°C and then I let it cool down on ice for 5min. After that I mixed the probe with fresh and 68°C Hyb solution.

Hope this helps...

Best regards,
Chakchel

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I am also using the same brand/type of membrane mentioned by Chakchel.
You mean you always use freshly prepared probe solution and didn't recycle it? What do you think about recycling the hyb solution? If you were to use the recycled hyb solution what would you do? Do you pre-heat it accordingly to the hyb temperature or you just thaw on ice? I am quite interested to know the answer as well. Thanks.

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Hi,

well, I think that reusing the Hyb Solution increases the possibilty of degeneration of your DNA the more often you use it... and also the background might increase because of degeneration of the Hyb solution itself...?
But I don't know... didn't try it. Sorry, I have no experience about reusing the Hyb solution.

But when I would do that, I certainly would preheat it.

I guess the probe can be reused (I heard it would be stable for some weeks), but you should boil and chill it again before use...

Are you going to try reusing the Hyb solution (maybe even more then once or twice)? Please let us know your results then - would be interesting for me, too.

Greetings,
Chakchel

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Is it a good idea to boil it and pre-heat it? The oligonucleotides that I am using is SS and I thought I can be just be used after thaw and pre-heated? (That's just my theory, any suggestion?) Wouldn't I destroying my probe if I boil the hyb solution? My pre-hyb and hyb solution contain 6xSSC, 16mMTris-HCl, 5x Denhardt's solution, 0.1% SDS and 75 microgramme/ml salmon sperm DNA. What do you think? Thanks for the help.

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Hi all...

I tried to reuse the Hyb soln and the good news is that it still worked. I didnt cool it down after heating it but straightaway poured it onto the membrane however I think that it would produce better yield if it was cooled down after heating before use. I was wondering whether the prehyb can be used? so far i've not heard anyone that reuses their prehyb, so i was wondering if anyone has ever tried that before.

Cheers,
Charlene

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