When trying to amplify a 700bp gene by PCR, a get a nouns of 1000bp. My primers are almost 60bp respectively. ?
It looks like you are getting non-specific products which method your reaction probably desires further optimization. Try one or some of the following:
- Decrease annealing time
- Increase annealing temperature
- Decrease extension time
- Decrease extension heat to 62-68o C
- Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.
- Increase MgCl2 concentration up to 3-4.5 mM but hold dNTP concentration constant.
- Take less primer
- Take smaller quantity DNA template
- Take less Taq polymerase
If none of the above works: check the primer for repetitive sequences (BLAST align the sequence beside the databases) and change the primer(s)
You're getting some non-specific binding. Try making the temperature more rigorous and perhaps smaller number cycles for the positive control and see if you get the right fastening. If not you may have to try a different set of primers. I other had to use two or three different primer combinations previously I got it right. Sure looks nice the first time you procure a nice clean PCR next to a single band contained by the right spot!