Think you're good at PCR? Try this one!
Bassuk at U.Washington.Edu
Sun Jan 15 21:04:17 EST 1995
In article <pemanuel-1401951420420001 at beaker.ab.umd.edu>, pemanuel at umabnet.ab.umd.edu (Peter) says:
>O.K. PCR Jocks. Try this one on for size...
>1. PCR amplifaction of IgG Heavy and Light chains yields a 700 bp PCR Fragment
>2. This fragment appears specific, is the correct size, and will
>re-amplify using an outside set of primers as is the case for the light
>3. The fragments are cut with Sfi (which is encoded in primer) and gel
>isolated with GENECLEAN.
>4. When this 700 bp fragment is run on a 1% gel to estimate the yield from
>the isolation a 1400 bp fragment suddenly appears with the original 700 bp
>5. Aha! You say-A dimer! Yes-heating the mixyure to 75°C before loading
>breaks it down to a 700 bp fragment. BUT..heating to 80-82°C gives a 700
>bp and a 450 bp fragment.
>6. So I figure the fragment is dimerizing. I mix the 2 frags and then
>heat them to disrupt the dimers and then proceed to ligate them together
>at the Sfi site. Lo and behold a 1400 bp fragment appears which doesn't
>seem have too much of those original dimers in them.
>7. I then cut the ligation mixes with Not I and Spe I and gel isolate the
>final 1400 bp fragment.
>8. When I run the fragment on a gel it appears to be mostly the 1400 bp
>fragment I want BUT when I heat it to 82°C it breaks up into the 700 bp
>fragments and this funny 450 bp fragment.
> So hear it is:
>??? Is this 450 bp fragment single stranded DNA?
> Why are these PCR fragments dimerizing? Sfi shouldn't allow that to happen.
> Is it possible that my 1400 bp fragment is good and that my heating
>test on the 1% agarose gel is simply denaturing it and it runs at what
>would appear to be a 700bp double stranded fragment?
>HELP I am desperate. What Am I doing wrong? What should I try next. The
>overlords are getting impatient.
My vote to solve this chap's dilemna would be to run your DNA in a gel
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