PCR help please

[shifalich from gmail.com]

On Sep 30, 11:32 pm, "Becky Pickin" <vpirei... from gmail.com> wrote:
> 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

Dear pickin!
Can you please tell, how you are making mix of dNTP again?
Shifali