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Need advice on a smear from primer-primer binding PCR reaction.


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#1 stagius24

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Posted 06 September 2010 - 06:23 PM

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.

#2 christy

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Posted 06 September 2010 - 08:34 PM

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.

#3 stagius24

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Posted 06 September 2010 - 08:45 PM

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




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