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[moheg]

My dear brothers,

i try to do ISSR Analysis for some Plant samples, bu there is no amplification after many trials
My protocol as the follows:

DNA extraction: by Promega Plant Dneasy Kit

and the pic of dna is attached

then i made Touchdown PCR as follows
25ul reaction:
H2O-------------------------9.875 ul
dNTPs (2mM) ----------------2.5ul
5x Green Gotaq buffer -------5ul
MgCl2 (25mM0 ---------------1.5ul
goTaq (5U/ul) ----------------0.125 ul
Issr Primer (10uM) ------------3 ul
DNA(conc. N/A) --------------3ul

Thermal Cycler program:
1) 95C --------------3 min
2) 95C---------------30 s.
3) (Tm+10) ---------45 s. (each cycle Temp. decrease -1C)
4) 72C --------------60 s.
go to (2) for 15 cycles
5) 95C -------------30 s.
6) (Tm ) -----------45 s
7) 72C -------------60 s.
go to (5) 20 cycles
8) 4C ---------------- Inf.

primer Tm= 51.780 (20mer)

And the pic after PCR is attached.

Any Idea to solve this problem ??
and, in 2nd pic, Is there is primer-Dimer?
many thanks for your efforts

Attached Files

•  DNA extraction.bmp   189.54K   18 downloads
•  Touchdown PCR.bmp   367.08K   16 downloads