PCR help please

[Becky Pickin]

Hello all,

I am back again this time asking for help with some problem PCR.  I will do
my best to explain what I have done.  If you have any suggestions or
tips/tricks I am quite willing to give them a try.  I have tested every
piece I can think of and have run out of ideas...none of my PCR reactions
have resulted in a band - specific or not.  All products should be between
200bp and 1200bp.  (negative controls are always blank - no streak, smear,
band..nothing - very clean)

Let me start with what I'm doing:

PCR on HeLa cell genomic DNA both EcoRI digested and not.  The initial
template used was <1 yr old HeLa genomic DNA isolated in our lab by another
member (excellent lab hands) and digested with EcoRI.  He has used this
template in PCR and for probe design within the past 6 months.
Concentration has been re-checked using a BioRad Spec measuring A260/280.
In addition, dilutions (down to the concentration used in PCR reactions) of
the DNA were run on a gel and resulted in the predicted smear (b/c it was
EcoRI digested). Alternative templates were freshly isolated (within the
past 2 weeks) HeLa genomic DNA not EcoRI digested and a purified plasmid
DNA.  All templates have been diluted to ~20ng/ul and 1ul added to the PCR
reaction.  Templates (except for the undigested genomic) have been used
successfully by other lab members.

I designed 7 pairs of primers using the Primer 3 program and double checking
primer dimers, etc. using the oligo calculator at Northwestern.  I have
experience designing primers and had another lab member (faculty) double
check them before I ordered the primers.  Primers are designed to have a Tm
of 55-65oC depending on how you calculate (ie salts, etc.)  The target Tm
was 60oC.  Chosen pairs were not anticipated to have any hairpins, primer
dimers, etc (I had enough flexibility to select against any that were
predicted to be problematic in that regards).  These are the "experimental"
primers.  All primers are reconstituted in TE at 100uM and then diluted to
10uM in autoclaved ddH2O for a working stock.  These were ordered and
reconstituted within the past 2 weeks.

Positive control primers are also at 10uM working stocks and have been shown
to give a band with the EcoRI digested HeLa genomic DNA within the week by
another lab member (as part of the trouble shooting process) in QPCR.
Dissociation curves show a specific product using Sybr Green chemistry.
These primers have also been used successfully by other lab members (using
more stringent reaction conditions, annealing temp of 60oC) and was why they
were selected as a positive control for these experiments.  I have been
unsuccessful at obtaining a product with these primers using any of the
three templates listed above.

My reaction chemistry is as follows:
                                                              * Conc in
reaction*     *Volume used (ul)/rxn*
10x PCR Buffer (-MgCl2) from Invitrogen
1x                            5
50mM MgCl2
1.5mM                     1.5
5u/ul Taq Polymerase
1u                            0.5
(these are all part of Invitrogen Platinum Taq Polymerase "kit" Cat
#10966-026 and are fresh and have been used in a PCR by another lab member
and resulted in a positive band)
Autoclaved ddH2O
-                              40
10mM dNTP mix                                             0.2mM
@                 1
Primer 1                                                         0.2uM
@                  0.5
Primer 2                                                         0.2uM
@                  0.5
Template (diluted to 20ng/ul)
20ng                   *     1       *

50ul per reaction

dNTPs are from Roche diagnostics (#11277049001) shipped as 100mM lithium
salt solutions at pH7 (10umol in 100ul).  I mixed the 4 dNTPs together as
such: 20ul of @dNTP + 120ul ddH2O, made within the past 2 weeks and stored
at -20oC.

Reactions are set up with a premix adding water followed by buffer first.
Some reactions had template added separately, some reactions had primers
added separately.


Reaction Conditions tried:
Try 1:
Step 1 94oC  2min
          57oC   1min      1 cycle
          72oC   2 min

Step 2  94oC  1min
           57oC   1min  28 cycles
           72oC   2min

Step 3  94oC  1min
          57oC   1min       1 cycle
           72oC   10min

Step 4  Hold at 4oC

Try 2:
Same as Try 1 with annealing temperature reduced to 55oC, total cycles
increased to 40

Try 3:
Step 1 94oC  2min
          55oC   1min    1 cycle
          72oC   2 min

Step 2  94oC  30 sec
           55oC   30 sec   38 cycles
           72oC   2min

Step 3  94oC  30 sec
           55oC   30 sec    1 cycle
           72oC   10min

Step 4  Hold at 4oC


2 different thermocyclers have been tried.  Both have a heated lid and thus
no oil was applied to the reactions.  PCR "products" have loading dye added
and then are applied to between 1% and 1.5% agarose gels.  The gels are run
at 100-120 volts until the dye front migrates ~2/3 of the way down the gel.
Gels are then stained with EtBr and photographed on UV light box.  Markers
used are beautiful.

Help please!

Thank you,
R Pickin, PhD