PCR help

C.S. Wilding BGYCSW at leeds.ac.uk
Mon Jun 8 03:08:35 EST 1998


I would have thought that the problem with this is that you 
will get non-specific priming early on and from then on the 
primers are incorporated. So whether you raise stringency 
gradually or not you will then be amplifying the wrong 
product under apparently stringent conditions. Increasing 
annealing temp. is therefore not used in most cases (although 
it sounds like it has worked for you) since you might as well 
just keep a low annealing temp- you will still amplify 
strongly the rubbish that was primed in the early cycles.
Craig.


In article <6lfrgh$r1q at mserv1.dl.ac.uk>,
   Alok Adholeya <aloka at teri.res.in> wrote:
>Dear All,
>
>
>I have a query on PCR fingerprinting. Conventionally during
>standardization of PCR we adjust the Mg++ concentration, 
standardize
>annealing time/ temperature, add specificity enhancers like 
formamide
>etc.... . I wrote a small programme for my REP on my PCR 
Machine.
>Instead of complete smears on my gel (which I have been 
getting) I
>ended up having distinct and beautiful finerprint. The 
programme is as
>following:
>
>#1 94 deg C for 2 min
>#2 94 deg C for 50 sec
>#3 35 deg C for 1:20 min
>     Increment of 0.5 deg C per cycle
>#4 72 deg C for 2:30 min
>#5 GoTo step number 2, 18 times
>#6 94 deg C for 50 sec
>#7 44 deg C for 1:20 min
>#8 72 deg C for 2:30 min
>#9 GoTo step number 6, 17 times
>#10 4 deg C for 5 min
>#11 END
>
>Rationale behind this programme? 
>We start with (very) low stringency as the Tm of both the 
REP primers in
>50 mM Na+ ions are 43 deg C thereby allowing even weaker 
binding
>sites have some access to the primer. As the reaction 
progresses the
>stringency is incresed (0.5 deg C per cycle) and within a 
few cycles
>(10) the annealing temperature comes close to Tm. After a 
while the
>majority of the reacton is carried out on extremely highly 
stringent primer
>annealing temperature (one deg above Tm). Extension slightly 
above Tm
>is possible from sties having very accurate match.
>The advantage of such a STEP-UP PCR (I call this so because 
it is
>analogous to Touch Down PCR) is that not a great deal of
>standardization is involved, no need to titrate Mg++ and 
other additives.
>
>Then what's the problem?
>I have not seen such a protocol for PCR fingerprinting, 
especially when
>the primer used has been extensively studied. Would people 
accept this
>kind of an amplification parameter. If not, why not? 
Afterall we are using
>the machine to enhance specificity rather than conventional 
PCR
>additives. I addend nothing except what was mandatory ie., 
Buffer,
>dNTP, primers, template Taq and water.
>
>Thanks
>Alok
>
>Please email me at  : aloka at teri.res.in
>



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