Help! Separating 2 PCR products.
qianfa at mgcheo.med.uottawa.ca
Thu Aug 28 10:45:26 EST 1997
Dharsha (mmm_dthurai at mednov1.auckland.ac.nz) wrote:
: I am trying to seperarte two PCR products, both 100-130 bp. They are
: specific for my 2 (20 and 16 mer) primers. As I am trying to get the
: Ig heavy chain rearragemnt (myeloma and ALL samples) PCRed up, it is
: not unsuprising that I should get two products closely related in
: I have tried to get just one band by:
: optimising MgCl concentration-still two bands.
: Increasing the temperature from "optimum" of 50 to 60 C-still two
: Hot Start PCR-still two bands
: Touch Down PCR-still two bands
: Primer Titration-still two bands
: The lower PCR band is ~120 and the upper band is ~130, so they are
: separate but not easily separable. I have tried cutting out and
: rePCRing the separate bands, but the lower band reappears. Using this
: I have tried to get the lower band sequenced.
: Please help me to try and get just a band form the PCR. Cutting the
: bands is not easy or really reproducible, and thus is not a good thing
: to use for screening our samples.
What about subcloning the mix of two bands into PCR2.1 (simple and easy) and you will
have 50% of the clones that carry one of the bands.
| Qianfa Chen Ph.D. qianfa at mgcheo.med.uottawa.ca
| ApoptoGen Inc. Tel: 613-738-3926
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