Diluted DNA for PCR???

Steve Hopkins shopkins at fs1.ho.man.ac.uk
Thu Jan 26 10:11:22 EST 1995


Re:
>I am trying to reamplify a DNA from a previous PCR, and for that I made
>a few dilutions to check which one works: 1ng/microliter, 0.1ng/", 100pg/",
>this last one gave me the best results, so I trued it again, it worked the
>same, but the 3rd time I ued it the bands were very faint, and the 4th time
>did'nt gave me any product at all. I am storing the dilution at -20, 
>defrozen it every time I used it. I was suggested that the DNA was stiking
>to the tubes and that by now there is no DNA in the solution, can this
>be possible, should'nt I detect something with the PCR???????

This might sond crazy, but we found that putting diluted PCR product back in 
to the RT cocktail (everything, though we can't see the RT itself is 
important) increases the sensitivity tremendously. We don't know why, and it 
works with hexamers, poly-A, or specific primer in the RT-mix, but if you try 
it please let me know. 

Steve Hopkins
Manchester



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