Need advice on a smear from primer-primer binding PCR reaction. - (Sep/06/2010 )
Hello,
I design 2 oligos around 80mer that will overlap each other at the end around 22bp.
Here is my reaction recipe
• PCR: 10X buffer 10ul
Lunasin_1-43F1(5pmol/l) 5ul
Lunasin_1-43R1(5pmol/l) 5ul
10mM dNTP mix 1ul
Taq pol (5u/l) 0.5ul
H2O 78.5ul
94C 30”
55C 30”
72C 30”
6 cycle
I tried many time, and always got a big smear below and above the band, there is very fainted unspecific band somewhere on top too.
Please give me advice. The oligos will be used later for cloning.
-stagius24-
stagius24 on Tue Sep 7 02:23:12 2010 said:
Hello,
I design 2 oligos around 80mer that will overlap each other at the end around 22bp.
Here is my reaction recipe
• PCR: 10X buffer 10ul
Lunasin_1-43F1(5pmol/l) 5ul
Lunasin_1-43R1(5pmol/l) 5ul
10mM dNTP mix 1ul
Taq pol (5u/l) 0.5ul
H2O 78.5ul
94C 30”
55C 30”
72C 30”
6 cycle
I tried many time, and always got a big smear below and above the band, there is very fainted unspecific band somewhere on top too.
Please give me advice. The oligos will be used later for cloning.
PCR reaction 95 10 mins, ur specific Tm based on ur primers (time depends on ur dna length) 72 same as ur Tm time and 4 degree hold.
usually, we design 15 mers for overlapping it is working well, ur is 20 mer, so it should work.
-christy-
why 95oC at 10 mins ? Can you explain
on the sheet, it said 1M NA tm is 120oC , 50nM NA is something 99oC .. I am not sure what is the real Tm
-stagius24-