IUBio

Help me! info about gene cloning -Reply

Steven Projan PROJANS at war.wyeth.com
Wed Feb 26 07:34:55 EST 1997


Il Minn writes:

" 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"

P:erhaps the approach that will work best for you would be to forget
about using a completely degenerate pair of primers and remember that
B. stearothermophilus has a very high GC content (and therefore a
clear bias in codon utilization).  So just use the most GC rich
codons for each amino acid, this should greatly limit the number of
primer possibilities.  You may also consider using your primers in a
Southern blot experiment - if you fail to get hybridization with your
primers then you will have difficulty getting the PCR to work.

Good luck and good cloning!

Steve Projan
Wyeth-Ayerst Research




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