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Forum: Sample Prep / Library Generation 07-22-2016, 03:20 AM
Replies: 2
Views: 907
Posted By firefly2280
Bacterial Transcriptome sample prep following different doses of drug

Dear all

If we wanted to look at bacterial transcriptomes and how they differ following exposure to different doses of a drug, how important isi t to keep the bacterial population the same? I...
Forum: Illumina/Solexa 02-13-2014, 07:52 AM
Replies: 23
Views: 24,569
Posted By firefly2280
Hi, I know I'm late to this conversation but...

Hi,

I know I'm late to this conversation but we had the same issues. I managed to 'borrow' some Indexed primers for V4 and for V5-7 regions and compare just a few on a small set of my samples. The...
Forum: General 10-06-2013, 02:51 AM
Replies: 6
Views: 3,478
Posted By firefly2280
Thats great, thanks do much for all of the...

Thats great, thanks do much for all of the replies!
Forum: General 09-30-2013, 12:26 PM
Replies: 6
Views: 3,478
Posted By firefly2280
Thanks. I was also looking for info regarding...

Thanks. I was also looking for info regarding sample prep, read-length too, that kind of thing but thanks for your link!
Forum: General 09-30-2013, 11:55 AM
Replies: 6
Views: 3,478
Posted By firefly2280
Does anyone have a comparison table of latest NGS tech?

Hi all

I was wondering if anyone had or knew a reference to a table comparing the brief specs of current platforms. It all changes so fast and I know there are some out there I know nothing about....
Forum: Sample Prep / Library Generation 09-30-2013, 11:48 AM
Replies: 4
Views: 3,247
Posted By firefly2280
Thanks again, I'll have a play with it again. I...

Thanks again, I'll have a play with it again. I dont see why I shouldnt be able to get it to work! At least I know Im not crazy and other people can make it work! Thanks
Forum: Sample Prep / Library Generation 09-29-2013, 11:10 PM
Replies: 4
Views: 3,247
Posted By firefly2280
Thanks for your reply. I tried that and got...

Thanks for your reply. I tried that and got nothing with the first primers after 10 cycles... Might have to give it another go. It works well with a full 30 cycles each but there's no way I want to...
Forum: Sample Prep / Library Generation 09-28-2013, 12:12 PM
Replies: 4
Views: 3,247
Posted By firefly2280
Struggling to amplify DNA with barcoded primers

Dear all

I am using the MiSeq for 16S amplicon sequencing with custom barcodes.

I use the barcoded primers on extracted genomic DNA. However, I have quite low concentrations of starting DNA,...
Forum: Illumina/Solexa 07-26-2013, 03:52 AM
Replies: 24
Views: 12,498
Posted By firefly2280
Phillip, Thanks so much, Im going to get someone...

Phillip, Thanks so much, Im going to get someone to help me take a look!

thanks again
Forum: Illumina/Solexa 07-23-2013, 12:13 PM
Replies: 10
Views: 2,914
Posted By firefly2280
Many thanks Number6. The primers are all being...

Many thanks Number6. The primers are all being used successfully by a colleague, so we dont think they are the issue. Thanks though
Forum: Illumina/Solexa 07-22-2013, 06:56 AM
Replies: 10
Views: 2,914
Posted By firefly2280
ok, thanks will do later. I dont have anything...

ok, thanks will do later. I dont have anything with me. Im really new to this (you can probably tell)!
Forum: Illumina/Solexa 07-22-2013, 06:43 AM
Replies: 10
Views: 2,914
Posted By firefly2280
Hi, sadly not as the runs failed. the samples...

Hi, sadly not as the runs failed. the samples failed to cluster, nothing worked. apart from the Phi X.
Forum: Illumina/Solexa 07-22-2013, 06:02 AM
Replies: 10
Views: 2,914
Posted By firefly2280
Thanks, they are the exact same ones as being...

Thanks, they are the exact same ones as being used by other MiSeq users in my lab and they are getting good results
Forum: Illumina/Solexa 07-22-2013, 05:23 AM
Replies: 10
Views: 2,914
Posted By firefly2280
The Phi X has clustered but the run then fails,...

The Phi X has clustered but the run then fails, so no reads at all from my samples.
Forum: Illumina/Solexa 07-22-2013, 04:50 AM
Replies: 10
Views: 2,914
Posted By firefly2280
Library failure - any help appreciated

Hi all

I am having trouble getting a MiSeq run to work and was hoping someone might be able to help

I have swab samples from surfaces in a hospital, i have DNA extracted using a bead beat and...
Forum: Illumina/Solexa 07-18-2013, 10:14 PM
Replies: 24
Views: 12,498
Posted By firefly2280
Thanks for your reply, yes, all ok. My colleague...

Thanks for your reply, yes, all ok. My colleague runs the same primers all the time and he helped me set up the run, so all was in order. It really seems sample related
Forum: Illumina/Solexa 07-18-2013, 05:04 AM
Replies: 24
Views: 12,498
Posted By firefly2280
Thanks, yes the primers were in the right wells...

Thanks, yes the primers were in the right wells and Im not using the experiment manager so I dont think that was it either.
Forum: Illumina/Solexa 07-18-2013, 05:02 AM
Replies: 24
Views: 12,498
Posted By firefly2280
I ran some samples on a collegues run which...

I ran some samples on a collegues run which worked but my samples only produced 4, 10 and 43 reads...!! so something is for sure going on!
Forum: Illumina/Solexa 07-18-2013, 05:01 AM
Replies: 24
Views: 12,498
Posted By firefly2280
Thanks for this. Im not using Nextera but I...

Thanks for this. Im not using Nextera but I suppose its 'good' to know that this is what might be causing a problem!
Forum: Illumina/Solexa 07-18-2013, 04:58 AM
Replies: 24
Views: 12,498
Posted By firefly2280
Thats what I think too but maybe its something...

Thats what I think too but maybe its something that associates with the DNA and co-purifies?
Forum: Illumina/Solexa 07-16-2013, 11:16 AM
Replies: 24
Views: 12,498
Posted By firefly2280
thanks for the input. Sadly Illumina are not...

thanks for the input. Sadly Illumina are not going to replace the kits and I cant afford to trial and error things, so I dont really know what to do now. The only thing any of us can think of for...
Forum: Illumina/Solexa 07-16-2013, 10:38 AM
Replies: 24
Views: 12,498
Posted By firefly2280
Thanks. Other people are doing successful runs,...

Thanks. Other people are doing successful runs, so I think it is a sample issue sadly!
Forum: Illumina/Solexa 07-16-2013, 06:24 AM
Replies: 24
Views: 12,498
Posted By firefly2280
The error message "Object reference not set...

The error message

"Object reference not set to an instance of an object”

came up and that was it, so didnt even get as far as cluster densities.

Illumina thought it was the concentration of...
Forum: Illumina/Solexa 07-16-2013, 04:16 AM
Replies: 24
Views: 12,498
Posted By firefly2280
MiSeq run failure - inhibition?

Hi all,

I recently did a MiSeq run that failed before it had really begun. I am doing 16S custom sequencing with the barcodes.

Everything was properly calculated, diluted and Qubited, so I am...
Forum: Sample Prep / Library Generation 11-05-2012, 04:35 AM
Replies: 12
Views: 12,000
Posted By firefly2280
Thanks for your help all. Nothing shows up...

Thanks for your help all.

Nothing shows up on a gel, im quite sure there are inhibitors, which is why i wanted to use a column based extraction method. I might switch back to phenol chloroform if...
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