Help me! info about gene cloning

Connie Hansen chansen at connectnet.com
Sun Mar 16 00:58:27 EST 1997


Il Minn wrote:
> 
>    I purified a protease, c.a. 80000Da, from Bacillus stearothermophilus
> and determined the N-terminal sequences for gene clonong. Based on that
> sequences I designed the oligonucleotide primer for PCR. I used the total
> genomic DNA as PCR template, but due to the high degeneracy of primer
> I could not get correct PCR product. So my questions are as follows
> 
> 1) Is there any method for increasing the specificity of PCR with highly
>    degenerate primer?
> 2) Is there any method for making cDNA from Bacillus RNA?
> 
> 3) Are there other methods for cloning a gene from Bacillus?
> 
>        I am very appreciate for your answers in advances
> 
>                                         Sincerely yours,
>                                         Il Minn
I had a lot of trouble with degenerate primers until I learned to keep
the degeneracy down to 100 fold and to use at least 500 ng of the
degenerate primer in the PCR reaction.  Once you reach 100 fold
degeneracy you have to grind your teeth and start ordering multiple
primers.  Expensive!



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