AMPure XP vs SPRI beads

[Tali7]

Hey guys

Curious as to which system you think is better? I've been trawling this forum regarding size selection using magnetic beads, for use in RADseq (a modified gDNA illumina prep). I'm just a little confused -

- can you buy SPRI beads separately from the technology/hardware? I can't find anything on the beckman page for just SPRI beads. Or am I being dumb and the beads rely on the hardware to work, so you have to buy the whole system for automated library prep?

- are AMPure XP beads (which I have access to) just as good at size selection as SPRI beads? I'm hearing a lot more about the SPRI system on this forum for size selection, rather than the XP beads.

Ideally I would like to size select for fragments 300-500bp, for which I'm aware I'll probably need to do a double selection (one to eradiate fragments >500bp, the other to eradicate fragments <300bp), which is fine.

Also, if anyone knows a good method to calculate the beadNA ratio other than trial and error, that would be greatly appreciated!

Cheers

Tali

[Chipper]

Ampure XP beads are SPRI beads. It is easy to test on a ladder, or look at the metagenomics paper in PloS one for the ratios.

[Tali7]

Ahh ok, I hadn't realised that! I'll try out a couple of ratios (based on that paper, thanks for that reference) on a ladder.

Thanks a lot

[xiaoyuan zi]

Originally Posted by Chipper

Ampure XP beads are SPRI beads. It is easy to test on a ladder, or look at the metagenomics paper in PloS one for the ratios.

I need the complete information of "the metagenomics paper in PloS one ". Thanks.

[xiaoyuan zi]

Originally Posted by Tali7

Ahh ok, I hadn't realised that! I'll try out a couple of ratios (based on that paper, thanks for that reference) on a ladder.

Thanks a lot

I want to know your result about the ratios. Thanks.

[Ber7702]

Hi Tali,
You are correct that the website is a little confusing! Our SPRI products are on the Beckman Coulter Genomics website. Here is a link directly through to the AMPure XP page.

https://www.beckmancoulter.com/wsrpo...tion/index.htm

If you have any further questions or queries then do not hesitate to get in touch.

Best wishes

[xiaoyuan zi]

Hi,

Thank you! I have the SPRI bead. I will use it to select PCR product of 300-500bp size. I use the recommend protocal but the cut-off is not clear. On the website I can not find any information. Can you help me?

[lucyyang1991]

hi, your link is wrong, there's no product information.
I'm just wondering if there is a way to search in your company website!

Originally Posted by Ber7702

Hi Tali,
You are correct that the website is a little confusing! Our SPRI products are on the Beckman Coulter Genomics website. Here is a link directly through to the AMPure XP page.

https://www.beckmancoulter.com/wsrpo...tion/index.htm

If you have any further questions or queries then do not hesitate to get in touch.

Best wishes

[Ber7702]

Dear LucyYang1991,
That is strange, I just clicked the link and it took me directly to the AMPureXP page. If it is your first time to the website, you may need to select your region and it should then take you to the product page.

You could try this link to our Life Sciences page.

https://www.beckmancoulter.com/wsrpo...very/index.htm

There is a small magnifying glass in the top right. Click on that to bring up the search bar.

[Ber7702]

Apologies, I was not notified that you had responded to my link. I realise this is now a very late reply but the cut off is around 120-150bp when using the standard x1.8 ratio.

It is not a sharp cutoff. You will still get some products smaller than the cutoff but with significantly reduced amounts. Below 100bp or so all the fragments will be removed.

Originally Posted by xiaoyuan zi

Hi,

Thank you! I have the SPRI bead. I will use it to select PCR product of 300-500bp size. I use the recommend protocal but the cut-off is not clear. On the website I can not find any information. Can you help me?

[lucyyang1991]

OK, thanks a lot, this is the first time to the website. Maybe that's the problem here.

Originally Posted by Ber7702

Dear LucyYang1991,
That is strange, I just clicked the link and it took me directly to the AMPureXP page. If it is your first time to the website, you may need to select your region and it should then take you to the product page.

You could try this link to our Life Sciences page.

https://www.beckmancoulter.com/wsrpo...very/index.htm

There is a small magnifying glass in the top right. Click on that to bring up the search bar.

[hssalgh2]

Originally Posted by Chipper

Ampure XP beads are SPRI beads. It is easy to test on a ladder, or look at the metagenomics paper in PloS one for the ratios.

hi
i am testing the ampure beads to see is it working or not on a ladder DNA but i got band in 100 pb and i did not understand it can you see my attached image!
how do we know if the Ampure beads working or not ?

thank you very much.
comparing beads 11.7.17.jpg

[Carcharodon]

Originally Posted by hssalgh2

hi
i am testing the ampure beads to see is it working or not on a ladder DNA but i got band in 100 pb and i did not understand it can you see my attached image!
how do we know if the Ampure beads working or not ?

thank you very much.
Attachment 4827

We would need to know what the conditions of your test were and which lanes correspond to which treatment before we could really answer your question. Could you tell us these things?

Also, from what I understand, it's better to test the beads on enzyme-digested lambda-DNA (to generate a known fragment size profile) rather than on pre-packaged DNA ladder. Something about an ingredient inhibiting binding/elution (I'm fuzzy on the details). This may or may not be true, however, so take it with a grain of salt.

[hssalgh2]

Originally Posted by Carcharodon

We would need to know what the conditions of your test were and which lanes correspond to which treatment before we could really answer your question. Could you tell us these things?

Also, from what I understand, it's better to test the beads on enzyme-digested lambda-DNA (to generate a known fragment size profile) rather than on pre-packaged DNA ladder. Something about an ingredient inhibiting binding/elution (I'm fuzzy on the details). This may or may not be true, however, so take it with a grain of salt.

Thanks for your reply and please see the attached Protocol that I had used for
Testing beads.
I use 2 microliter of ladder DNA diluted with 18 microliter of dearer and I add 36 microliter of beads then I complete the steps that in the attached file.
Many thanks

[Carcharodon]

Originally Posted by hssalgh2

Thanks for your reply and please see the attached Protocol that I had used for
Testing beads.
I use 2 microliter of ladder DNA diluted with 18 microliter of dearer and I add 36 microliter of beads then I complete the steps that in the attached file.
Many thanks

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).

[hssalgh2]

Originally Posted by Carcharodon

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]

Originally Posted by hssalgh2

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]

Originally Posted by Carcharodon

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]

Originally Posted by hssalgh2

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]

Originally Posted by hssalgh2

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.