NEED PROTOCOL: PCRing genes from E.Coli

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Mon Jul 3 22:28:11 EST 2000


what exactly do you need by way of a protocol? The technique of PCR is such
a routine these days that any basic mol. bio handbook should have it.

Assuming that you have the proper primers and all the reagents you need for
a successful reaction, all you really need to do is grow up a mL of the E.
coli cells in LB, wash the cells in TE (pH 8) briefly and lyse the cells by
hypotonic shock using clean water and boiling. Alternatively, you can add
an aliquote of the resuspended cells to the PCR reaction directly and start
the cycles.

Assuming you are absolutely a first-timer:

1. Read up a protocol from either the Red or the Blue book.

2. You need two primers ("facing" each other) complementary to the
extremities of the target seq. Lengths between 15 to 28 usually works in
most situations. Usually a pair of G's or C's or GC at the 3' end of the
primer ensures stability of primer ammealing (I don't care about the
sporadic controversies re this:)). Balanced GC-AT in the primer seq. also
is desirable, but depending on the target this ratio may be slightly
skewed, provided the primer pairs anneal within 5 degrees of each other. It
also helps to keep the Td between 45 and 55 degrees.

3. All the reagents needed are available commercially, including the water!

4. Since the E. coli genome sequence is known, it may be helpful to digest
the genomic DNA at an appropriate site(s) to avoid non-specific amplification.

At 06:12 PM 7/3/00 GMT, replace no-spam with s691461 to reply wrote:
>
>Can someone give a working protocol 
>
>for PCRing a gene from E.Coli genome?
>
>
>
>Thanks in advance!
>
>Anton.
>
>
>
>
Dr. Hiranya Sankar Roychowdhury
Dept. of Molecular Biology	
PO Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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