Hi everybody
I am new here and would like to first say thank you for this great forum. I'm looking forward to being active in it.
I have a problem with emPCR lib L. Everything worked fine until the very last step, the sequencing primer annealing. All our heating blocks were occupied so I used one of another group. I had problems with checking the temperature and it is possible that the temperature hadn't reached 65°C yet when I put in my sample. At the end I figured it out and the temperature was right, but it's possible that it was only on 55°C or so at the beginning.
Now I'm afraid that the primers may have bound nonspecifically or that it was only for some 2-3 minutes at 65°C and not enough primers could bind to the DNA.
I would have enough sequencing primers for another annealing round. Do you think I could add some melting solution to remove all sequencing primers and the perform washes with annealing buffer and then again perform the sequencing primer annealing step?
I really hope you can give me some advice soon. Thanks in advance.
Cheers,
Isabel
I am new here and would like to first say thank you for this great forum. I'm looking forward to being active in it.
I have a problem with emPCR lib L. Everything worked fine until the very last step, the sequencing primer annealing. All our heating blocks were occupied so I used one of another group. I had problems with checking the temperature and it is possible that the temperature hadn't reached 65°C yet when I put in my sample. At the end I figured it out and the temperature was right, but it's possible that it was only on 55°C or so at the beginning.
Now I'm afraid that the primers may have bound nonspecifically or that it was only for some 2-3 minutes at 65°C and not enough primers could bind to the DNA.
I would have enough sequencing primers for another annealing round. Do you think I could add some melting solution to remove all sequencing primers and the perform washes with annealing buffer and then again perform the sequencing primer annealing step?
I really hope you can give me some advice soon. Thanks in advance.
Cheers,
Isabel
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