[hssalgh2]

I see! Well, I'd recommend using a ratio slightly less than 1.8x. I'd lower that to 1.5x or so and see if it makes a difference (30 uL AmpureXP to 20 uL ladder dilution).

Thanks. I have chosen this ratio because I am planning to do 16s rRNA amplicon sequencing so I have 96 samples and I will do nested PCR. So what do you think about this ?
Many thanks

[Carcharodon]

Thanks. I have chosen this ratio because I am planning to do 16s rRNA amplicon sequencing so I have 96 samples and I will do nested PCR. So what do you think about this ?
Many thanks

I don't think it should be much of a problem, unless I'm misunderstanding something. If the lower ratio is a concern, I would recommend taking one (or a few) of your PCR products and trying both ratios on them, running them out on a gel.

So on the gel you would run:

1) PCR Product w/o cleanup
2) PCR Product w/ cleanup at 1.8x
3) PCR Product w/ cleanup at 1.5x

...and see if there's a difference.

It's a bit of extra work, but it's a small price to pay for peace of mind. This approach may also be better than testing on a ladder, since you'll be working with the PCR products directly (rather than with a ladder which may or may not have an inhibitory effect on the SPRI beads).

[hssalgh2]

I don't think it should be much of a problem, unless I'm misunderstanding something. If the lower ratio is a concern, I would recommend taking one (or a few) of your PCR products and trying both ratios on them, running them out on a gel.

So on the gel you would run:

1) PCR Product w/o cleanup
2) PCR Product w/ cleanup at 1.8x
3) PCR Product w/ cleanup at 1.5x

...and see if there's a difference.

It's a bit of extra work, but it's a small price to pay for peace of mind. This approach may also be better than testing on a ladder, since you'll be working with the PCR products directly (rather than with a ladder which may or may not have an inhibitory effect on the SPRI beads).

Ok. I will do waht you suggested. Then I see
I am thinking about PCR cycling for the first round is 18 cycle and in the second round is25cycle so is it ok to increase the cycle in the first round to 35 cycle what do you suggest?
Many thanks

[Carcharodon]

Ok. I will do waht you suggested. Then I see
I am thinking about PCR cycling for the first round is 18 cycle and in the second round is25cycle so is it ok to increase the cycle in the first round to 35 cycle what do you suggest?
Many thanks

This is probably better left for someone else to answer. I don't have any real experience with this protocol. I've gone toe-to-toe with the beads more than a few times, but nested PCR is another story.

[nucacidhunter]

Ok. I will do waht you suggested. Then I see
I am thinking about PCR cycling for the first round is 18 cycle and in the second round is25cycle so is it ok to increase the cycle in the first round to 35 cycle what do you suggest?
Many thanks

It depends on your experiment and input. It is not clear if you are preparing 16S region specific library for sequencing on Illumina systems or whole 16S for long read sequencing.