Problem with PCR of 2 kb linearised plasmid.

Kevin Morano ez005528 at rocky.ucdavis.edu
Mon Aug 30 14:39:03 EST 1993


mybdav02 at uctvax.uct.ac.za wrote:
: Hi folks

: I'm having some problems trying to PCR up a 2 kb plasmid (pWM521).  I have
: linearised the plasmid with SmaI and then designed primers for the ends
: that have been created.  The primers are 25 bases long, and have an 11
: base overhang (the overhang contains unique restriction sites).  Their Tm's
: are around 67 deg.

: I user the following conditions for my PCR:

:    92 deg  2 min
:    92 deg  45 s )
:    60 deg  60 s ) cycle 30x
:    72 deg  90 s )
:    72 deg  5 min

:   0.2 uM primers
:   30 ng  template
:   1.5 mM MgCl2
:   0.2 mM dNTPs
:   Promega 10x Taq buffer OR special buffer containing 300 mM Tricine
:                                                       0.1 % gelatin
:                                                       1 % thesit
:                                                       50 mM mercaptoethanol
:   2.5 U Taq
:   Water to 50 ul
:   50 ul paraffin to cover solution

:   Using these conditions, I get three bands on a 1% agarose gel.  The top 
: band is the correct length, and the lower two correspond to unrelated lengths.
: I have tried increasing the annealing temp to 65 deg to reduce non-specific
: binding of the primers, but this results in a decrease of the desired product.

: Is there something that I am doing wrong??  I've checked all my calculations
: many times, as have various people in my lab.  I have a bad feeling that the
: bands are due to binding elsewhere in the plasmid that is tighter than at the
: desired position.  

: If anyone has any ideas, please let me know - I'm getting fairly desperate as
: I have to get my insert cloned by the end of September (the insert has already
: been amplified by PCR - its conditions also don't work for pWM521).

: Thanks in advance

: Dave

: Dave Myburgh
: Dept of Biochemistry
: University of Cape Town
: mybdav02 at uctvax.uct.ac.za

Dave- Call up the molbio database at ftp.bio.indiana.edu and get the
program Amplify by Bill Engels. It will predict your secondary bands using
known sequences and primers and is VERY good. It has identified many bands
for us and helped eliminate potentially disastrous rxns.

One other note- I have had great success PCRing from crappy miniprep (alk.
lysis) DNA. I will do a 1 ml mp, r/s DNA in 40 ul and PCR using 0.5 ul of
this template in 100 ul rxn volume. It has yet to fail to amplify bands 2
kb or less. Might work for you.


--
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Internet:kamorano at ucdavis.edu     "Why does it happen, because it happens..."
Bitnet:kamorano at ucdavis                                              N. Peart
Section of Microbiology, University of California, Davis



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