Problem with PCR
smori at nmsu.edu
Thu Dec 22 16:22:28 EST 1994
Varada P. Rao Memorial Univ. of Newfoundland St. John's Canada (vprao at kean.ucs.mun.ca) wrote:
: Hi netters,
: I have experienced of late a new problem in addition to the weak signals
: that I often get in my PCR. I perform PCR to amplify T cell receptor cDNA
: using several Vb-specific primers but using a single constant region primer.
: A year ago I have done initially some PCRs to deterimine optimal conditions
: for my work. After that, I recently started again to repeat the previous
: observations. All the reagents and primeres were stored in -20 Celcius.
: Here comes the problem:
: I do get the appropriate sized bands specifically ( negative controls do
: work) but now, with almost all the appropriate Vb-primers + the same
: constant primer, I get " doublet-bands"; one of the correct size and
: the second one is smaller by 50bp approx. This pattern is constant in every
: Vb-specific PCR.
: This double-band problem is never seen with beta-actin primers under
: identical conditions.
: I am really going mad with this. I would greatly appreciate any input or
: Thanks very much in advance.
: "vprao at kean.ucs.mun.ca"
Sounds like your primers are not completely gene specific.There might be sites
that the primers are binding to. From the specificity of your product it seems
that may be the problem.
Maybe changing your primers would help.
Shahram Mori _/\_
Program in Molecular Biology _\ /_ Saskatoon/SK/CANADA
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
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