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
