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Northern bolt problem


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3 replies to this topic

#1 fox97

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Posted 02 April 2002 - 03:46 PM

Hello,

Recently, i always got high background low signal norther blot. The signal was very easy to washed away (10 min washing at 60 degree)  The protocol was same as i used before. But i had no problem of the northern blot before.

The diffences were

1) i made the probe again because the old one was used up.

2) i bought new membrane with same catalog number but different lot number

At first i though it might be the probe problem. But i checked the probe on agarose gel. The size and concentration were right. No degradation was seen from the gel.

When i used GAPDH to do the northen blot, it gave me very good result.

Can anybody here tell me the possible reasons?

Thanks a lot



#2 franck

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Posted 03 April 2002 - 06:51 AM

Hye

Have change anything in a buffer? or in the stockage of your buffer for northern?
And your probe is clean? (no possible degradation)


#3 milanoj

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Posted 04 April 2002 - 04:04 AM

It does sound like your probe is dirty. There may be unincorporated radionuclide in the mixture which will give high background and obscure the signal from the probe.

#4 fox97

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Posted 04 April 2002 - 04:51 AM

Thanks a lot: Franck and milanoj.

I used the new dextran sulfate in the hybridation buffer. The MW was 50,000. I think it's OK.


For the probe, this is how i made it:

1) enzyme digestion from plasmid DNA
2) separate on agarose gel
3) cut the 700bp probe fragment from gel
4) purify the probe from the gel using GENE Clean II from Bio101

when  chcked the probe by agarose gel, it showed a clear and sharp band at 700bp.  Is it possible that there r some  comtaminations that  can't be detected by agarose gel?

After labeling, i used G-50 quick spin column from Boehringer Mannheim to separate the labled probe from 32P-dCTP. It should be OK.

thank u very much






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