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Forum: Sample Prep / Library Generation 12-06-2013, 06:05 AM
Replies: 28
Views: 9,872
Posted By addyblanch
The KAPA kit is a SYBR green mixture.

The KAPA kit is a SYBR green mixture.
Forum: Illumina/Solexa 11-27-2013, 07:28 AM
Replies: 10
Views: 5,131
Posted By addyblanch
Are you trying to do something like TraDIS? We...

Are you trying to do something like TraDIS? We had a go and ended up using abridged illumina primers for it to work successfully. But even with this when quantified using bioanyliser they were all...
Forum: Illumina/Solexa 11-27-2013, 07:24 AM
Replies: 9
Views: 3,582
Posted By addyblanch
+1 for KAPA kits

+1 for KAPA kits
Forum: Ion Torrent 09-05-2013, 09:49 AM
Replies: 19
Views: 5,161
Posted By addyblanch
Your ng/ul will be different pre and post clean...

Your ng/ul will be different pre and post clean up because of the volume difference. You have a 20ul PCR reaction and a 25ul clean up elution.

PCR won't amplify the same with different primers!
Forum: Ion Torrent 09-05-2013, 08:50 AM
Replies: 19
Views: 5,161
Posted By addyblanch
Also you said your pre/post clean up volumes were...

Also you said your pre/post clean up volumes were the same you're eluting into 25ul. 5ul more than the PCR reaction. This would account for a ng/ul concentration difference but not total. The minimum...
Forum: Ion Torrent 09-05-2013, 08:45 AM
Replies: 19
Views: 5,161
Posted By addyblanch
If you can post the concentrations before and...

If you can post the concentrations before and after clean up.
Forum: Ion Torrent 09-05-2013, 08:37 AM
Replies: 19
Views: 5,161
Posted By addyblanch
So 40ng per reaction. I'd stop worrying about the...

So 40ng per reaction. I'd stop worrying about the beads. Its more likely the PCR. To be honest you're not getting great amplification from any reactions.

Take sample 5 in gene B for example if you...
Forum: Ion Torrent 09-05-2013, 08:20 AM
Replies: 19
Views: 5,161
Posted By addyblanch
What volume of 8ng/ul DNA have you used in the...

What volume of 8ng/ul DNA have you used in the reaction?

I doubt this is an SPRI bead issue. It sounds like a problem with your PCR. Have you run a positive and negative control? It might be an...
Forum: Ion Torrent 09-05-2013, 08:09 AM
Replies: 19
Views: 5,161
Posted By addyblanch
I thought it was an issue of concentration before...

I thought it was an issue of concentration before and after a SPRI clean up?

If its a question about 2 separate PCR reactions it might be that you are not amplifying anything in your second...
Forum: Ion Torrent 09-05-2013, 07:59 AM
Replies: 19
Views: 5,161
Posted By addyblanch
The table isn't that easy to follow, but from...

The table isn't that easy to follow, but from what I can tell is you have lost DNA. Quite a lot in fact. 17ng/ul to 0.4ng/ul is significant. It might be a faulty batch of SPRI beads. We had a similar...
Forum: Service Providers 08-30-2013, 05:31 AM
Replies: 14
Views: 9,189
Posted By addyblanch
ADAC at the University of Nottingham? ...

ADAC at the University of Nottingham?

http://www.nottingham.ac.uk/adac/index.aspx
Forum: Sample Prep / Library Generation 08-30-2013, 12:47 AM
Replies: 1
Views: 4,391
Posted By addyblanch
I don't see why not. I'd trial it first on a...

I don't see why not. I'd trial it first on a non-precious sample just to be certain. I prefer the NEB kits to Illumina kits and have always had better results in comparison.
Forum: Ion Torrent 08-30-2013, 12:38 AM
Replies: 19
Views: 5,161
Posted By addyblanch
Be aware that temperature of the Qubit reagents...

Be aware that temperature of the Qubit reagents can alter results. So if you kept the tube in the Qubit housing or even in your hand longer or at all, it could cause a change. Also if you made up the...
Forum: Sample Prep / Library Generation 08-28-2013, 03:06 AM
Replies: 8
Views: 6,817
Posted By addyblanch
If you're not looking to size select at this...

If you're not looking to size select at this point why not up your SPRI bead concentration to the recommended 1.8x? This might help with recovery.

If 0.7x removes fragments ~300bp your product at...
Forum: Metagenomics 08-14-2013, 02:33 AM
Replies: 9
Views: 5,914
Posted By addyblanch
Could look at KAPA HI-FI? ...

Could look at KAPA HI-FI?

http://www.kapabiosystems.com/products/name/kapa-hifi-pcr-kits

They have become NGS library prep gold standard very rapidly because of their low error rate and...
Forum: Sample Prep / Library Generation 07-02-2013, 12:23 AM
Replies: 2
Views: 2,491
Posted By addyblanch
Qubit Quantification

Hi All,

I've been using Qubit dsDNA kits to quantify my DNA preps for about 12 months.

Recently we've had issues with low yields from our preps so to save Qubit reagents while troubleshooting...
Forum: Sample Prep / Library Generation 06-11-2013, 02:54 AM
Replies: 10
Views: 1,676
Posted By addyblanch
Just to add, settings are different depending on...

Just to add, settings are different depending on which covaris you use.
Forum: Illumina/Solexa 06-04-2013, 05:49 AM
Replies: 4
Views: 5,070
Posted By addyblanch
Hi Phillip, I haven't denatured the...

Hi Phillip,

I haven't denatured the adaptors, I had read in a troubleshooting guide that a 70 hold was needed to ensure no secondary artefacts are present. I had miss understood and this is...
Forum: Illumina/Solexa 05-28-2013, 05:52 AM
Replies: 4
Views: 5,070
Posted By addyblanch
Unhappy TruSeq PCR Free LT Kit Issues

Hi all would appreciate any input on an issue i'm having.

I've moved across to the TruSeq PCR free kit and seem to be struggling with generating a library.

I'm fragmenting by covaris to 550bp...
Forum: Illumina/Solexa 02-28-2013, 04:42 AM
Replies: 7
Views: 3,461
Posted By addyblanch
Hi Scott, I'm in the process of designing...

Hi Scott,

I'm in the process of designing one with a collaborator. I would be interested to hear what you have done.

So far I'm aware that you can use custom primers in the empty wells on the...
Forum: Illumina/Solexa 02-11-2013, 02:49 AM
Replies: 13
Views: 3,151
Posted By addyblanch
Just thought i'd add something from my...

Just thought i'd add something from my experience. I too found this drop in quantity but found it not to be the fault of the covaris but of the qiagen kit.
After moving to a SPRI bead clean up the...
Forum: Sample Prep / Library Generation 01-28-2013, 04:52 AM
Replies: 2
Views: 2,352
Posted By addyblanch
Illumina Nextera Enrichment Buffers

Hi Everyone,

Just a quick question.

I'm currently trying to enrich a library prep using solution hybrid capture. I'm following the Nextera Enrichment protocol however because i'm studying...
Forum: Illumina/Solexa 01-24-2013, 12:39 AM
Replies: 1
Views: 1,491
Posted By addyblanch
Hi, I'm not too sure about the specifics of...

Hi,

I'm not too sure about the specifics of an ocogene panel, but if it helps PhiX is useful for low complexity samples. For this you can use between 30-40% but as internal control you can use 1%...
Forum: Sample Prep / Library Generation 01-15-2013, 02:50 AM
Replies: 3
Views: 4,755
Posted By addyblanch
I think they look correct, the movement in the...

I think they look correct, the movement in the "shoulder" would just be down to the amount of fragments at that size. They'll never all be the same but as long as the average size is your target,...
Forum: Sample Prep / Library Generation 10-11-2012, 02:36 AM
Replies: 2
Views: 1,417
Posted By addyblanch
Thats great thanks!

Thats great thanks!
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