Hi All,
I have a question about "Auto-sticky" PCR [1]
[1] Gal, J., Schnell, R., Szekeres, S. & Kalman, M. Directional cloning
of native PCR products with preformed sticky ends (Autosticky PCR). Mol.
Gen. Genet. 260, 569-573 (1999).
It works well before, the primers are:
Forward: /5Phos/TTG AAA TAC CGA CCG CTC AGC TAT CAG CC -- /idSp/ -- CTC
TGA CAC ATG CAG CTC CC
backward: /5DigN/ CAA CAA CGT TGC GCA AAC T
The primers were ordered from IDT, and "/idSp/" in FWD means abasic
site. The sequence after /idSp/ in FWD "CTC TGA CAC ATG CAG CTC CC" was
the real PWD oligo. As the presence of /idSp/, the first part
"/5Phos/TTG AAA TAC CGA CCG CTC AGC TAT CAG CC" would stay as a 5'
overhang. That was the basic idea of "Auto-sticky" PCR [1].
For PCR, we used Phusion Master Mix with HF Buffer (F-531L from
FINNZYMES). The cycling is
(1) Initial denaturation 98 C 30 s 1 cycle
(2) Denaturation 98 C 10 s 34 cycles
Annealing 59 C 30 s
Extension 72 C 23 s (15 s/1 kb)
(3) Final extension 72 C 10 min 1 cycle
4 C hold.
This protocol works fine, However, recently, we changed the FWD primer:
/5Phos/GGG CGG CGG GCG GGC GGG CGG GCG GGC GG -- /idSp/ -- CTC TGA CAC
ATG CAG CTC CC . There are many GC in the first part, and we thought it
will not change PCR as the second PCR primer after "/idSp" were same as
before.
But after run many times, there are no any PCR product. Does anyone can
give some suggestion?
Thank you so much and happy new year!
Zhi Qi
Biophysics at UIUC
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