Hi everyone,

Why could the band appear smudged?

I have amplified a 300 bp DNA G-block using Q5 2X mastermix (NEB) for 34 cycles, using 10-uM primers and 1 ng of template G-block as per protocol (M0492S). The primers generate overhangs and are as follows: fwd: Tm=47'C, 20mer, 35% GC, rev: Tm=59'C, 8%, 148mer, 8% GC. My colleague ran gradient PCR to determine best annealing temp of 57'C.

Gel electrophoresis: 1% TAE agarose gel, ran at 120V for 30 min and 90V for 15 min at 400mA. Ladder: 1Kb Plus DNA ladder (NEB)

PCR regime: 98'C:30 sec initial denat., 34X[98'C:10sec, 57'C:30 sec, 72'C: 20 sec], 72'C: 2 min final extension, 4'C hold.

Lane1: ladder

Lane2: 20 uL of PCR rxn+3 uL loading dye,

Lane3: 20 uL -ve control (no template) + 3uL loading dye.

I am wondering if:

1) I added too much PCR rxn per gel?

2) Gel concentration is too low?

3) Rev primer is way too large and formed dimers

4) Ladder did not separate too well so it is unclear what the MW of the smudge is

5) Annealing temp is not suitable?

6) Extension time too long?

7) Running buffer needs to be changed?

8) Voltage is too high?

9) Too many PCR cycles?

I would appreciate any feedback!

Thanks,

Maria