All gradient samples give bands! How come? - (Jul/11/2009 )
I just received new primers so i ran a gradient PCR to determine best annealing temp.
surprisingly all of my 5 tubes give the correct band.
temps were 57, 59, 61, 63 and 65C.
the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer
-Curtis-
Its Okay
The smear could be coz of high template, just cut yr band of interest and proceed.
Best,
TC
Curtis on Jul 11 2009, 08:43 PM said:
I just received new primers so i ran a gradient PCR to determine best annealing temp.
surprisingly all of my 5 tubes give the correct band.
temps were 57, 59, 61, 63 and 65C.
the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer
surprisingly all of my 5 tubes give the correct band.
temps were 57, 59, 61, 63 and 65C.
the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer
-T C-
The one that gives the brightest band is the one with the good annealing temperature. your purpose is to find the optimal annealing temperature right?
-jiajia1987-
Try reducing the amount of DNA you're using
do 1:10 and 1:100.
see if it helps
-hanming86-
Curtis on Jul 11 2009, 11:13 PM said:
I just received new primers so i ran a gradient PCR to determine best annealing temp.
surprisingly all of my 5 tubes give the correct band.
temps were 57, 59, 61, 63 and 65C.
the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer
surprisingly all of my 5 tubes give the correct band.
temps were 57, 59, 61, 63 and 65C.
the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer
This happen to me before. what I do was reduce the annealing time and extension time, and the smear gone, at least it works for me.
Hope you are not running a "rush" electrophoresis (160w ??)...this might make the smear on top happen too...for my experience.
-adrian kohsf-