ask for an explanation to pcr ladder at high cycle number - help (Dec/16/2005 )
Hi, dear colleagues,
I am doing rt-pcr on cadherin, integrin-beta1, and connexin.
The bands of these appeared at right position in the electrophoresis gel as pcr cycle number set at 18,23,25,and 30. However, as the cycle number increased to 35, all the three lanes showed ladders in the gel, and the last band of the ladder located at the right bp position.
Who could help me to give this phenomenon an explanation?
Thanks in advance.
-rickyama-
DNA can fragment and self prime - ie if it is left for too many cycles, the DNA fragments and acts as a primer and starts expending - hence u have DNA products larger than your target.
That said this should not be seen at 35 cycles? Perhaps your taq just does not last long enough?
-janbrisbane-
QUOTE (janbrisbane @ Dec 23 2005, 12:13 AM)
DNA can fragment and self prime - ie if it is left for too many cycles, the DNA fragments and acts as a primer and starts expending - hence u have DNA products larger than your target.
That said this should not be seen at 35 cycles? Perhaps your taq just does not last long enough?
That said this should not be seen at 35 cycles? Perhaps your taq just does not last long enough?
if u only want to see the presence of the band then the smaller number of cycles shud be enuff....larger number of cycles can lead to appearance of non-specific bands which probably do not get amplified in the smaller number of cycles as much to show up as bands
-Watson-