Hello, I did inserts:
In article <85nljf$5a7$1 at nnrp1.deja.com>,
ed_taboada at hotmail.com wrote:
> Hi everyone,
>> I'll be doing asymmetric PCR to generate a single stranded product which
> we will want to quantify. My boss worries about the amount of double
> stranded product that will be created and would like to quantify the
> amount of each species that will be generated by the PCR. We need to
Make sure the template is free of primers, if it was created by pcr: any of
the silica matrix kits are great (HighPure). If there are free phosphates,
as in a pcr, do an alk phosphatase to destroy any ability for extension:
clean w. the silica matrix to get into your required buffer (can be
simultaneous if template is scarce, but best results in correct buffer). The
same is also true for plasmid, cosmid, phagemid, etc. dna. In this case it is
a good idea because nonspecific contamnation is cleaned up as well.
> avoid double stranded product like the plague and are even considering
> cleaning-up the product by using a biotinylated primer/streptavidin
> magnetic beads/denaturation. We want to start with a primarily ss
> product (hence the asymmetric PCR) in order to minimize the costs
> associated with the (outrageously expensive) magnetic beads.
Following the asymetric pcr perhaps you could try using restriction enzymes
to cleave the ds and then isolate by size. How will you quantify the
product? In combination with anchored primers, and a cocktail of restricion
enzymes for outside of the primer sequence, you would likely destroy full
length ds. Without using anchored primers, digest followed with an alk phos
step in conjunction with silica clean would give you full length ss only for
template and get rid of priming from the ds. I have used this for cloning
only, not quantitation.
>> What sort of primer ratios might allow significant generation of ss
> product while minimizing the relative amount of ds product ?
The design of the asymetric pcr amplification primer is of greatest
importance. Choose the template dna binding region having the least homology
in comparison to the reverse 3' end of the amplified ss dna, and balancing
binding temp of >72C (2AT + 4GC) . Keep the primer as short as possible, and
avoid hairpin structures like in restriction sites (see above). Standard
primer conditions for the single primer should work (~20microMolar).
>> Also, is there a way for us to quantify the amounts of ssDNA and dsDNA ?
Do a test pcr to see what poor conditions (low binding temp, high cycles)
create. Run out the sample to see what the final result looks like, and run
ds linearized that is the size of the expected ss product. SS looks bigger
and dimmer than ds. I am sure you could find a respective quantization
procedure, but I don't know it.
>> If anyone could point me in the right direction I would be most
>> thanks in advance,
>> Sent via Deja.com http://www.deja.com/>
Happy New Year, Warren
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