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Forum: Illumina/Solexa 11-19-2014, 02:29 AM
Replies: 16
Views: 10,306
Posted By mikaelk
Hi, I would recommend to gel purify your...

Hi,

I would recommend to gel purify your samples before starting the library construction AND after the PCR. I used for the first gel a ladder called ZR small RNA ladder from Zymo to correctly...
Forum: Vendor Forum 12-05-2013, 02:09 AM
Replies: 6
Views: 4,673
Posted By mikaelk
Hi, I like the way the kit is designed. I...

Hi,

I like the way the kit is designed.
I was wondering if there is any chance to make the kit work with bacterial RNA, so without the polyA tail constrain ?

Thank you very much,

Mikael.
Forum: RNA Sequencing 08-01-2012, 07:18 AM
Replies: 11
Views: 5,993
Posted By mikaelk
Yes with the amounts mentioned above.

Yes with the amounts mentioned above.
Forum: Sample Prep / Library Generation 07-30-2012, 06:57 AM
Replies: 2
Views: 1,985
Posted By mikaelk
No I don't. It depends a lot on your sequencing...

No I don't.
It depends a lot on your sequencing requirements.
If you do need a lot of coverage (so reads) on mRNA, keep the lowest thershold that you can.
If having a high coverage on transcripts...
Forum: Sample Prep / Library Generation 07-30-2012, 06:48 AM
Replies: 1
Views: 904
Posted By mikaelk
I have not used the mirvana kit but there is one...

I have not used the mirvana kit but there is one thing that I know for sure.
If you do not see any ribosomal peak, it does not mean that you don't have any rRNA. You might came down from 95% of rRNA...
Forum: Sample Prep / Library Generation 07-30-2012, 06:26 AM
Replies: 2
Views: 1,298
Posted By mikaelk
As far as I know, the samples should be heat...

As far as I know, the samples should be heat denatured (2mn at 70C) prior to loading on the chip.
Once denatured, the samples are kept on ice until loading. This is what is specified in the user...
Forum: RNA Sequencing 07-27-2012, 06:57 AM
Replies: 11
Views: 5,993
Posted By mikaelk
Hi, Well, it depends a little on what you...

Hi,

Well, it depends a little on what you exactely intend to do on what organism.
I have get succesfull libraries with the protocol without any modification starting with 7L of 12000pM (so 84...
Forum: RNA Sequencing 06-11-2012, 09:11 AM
Replies: 11
Views: 5,993
Posted By mikaelk
I can only agree with this. smallRNA libraries...

I can only agree with this. smallRNA libraries are difficult to do and I do not recomment starting with totRNA.
In my experience (but I don't know the organism you are working with), the advised...
Forum: RNA Sequencing 06-11-2012, 08:58 AM
Replies: 1
Views: 1,153
Posted By mikaelk
Hi, As far as I know having barcodes ligated...

Hi,

As far as I know having barcodes ligated directly to the fragmented RNA is a bad idea and I would not recommend that.
The ligase is not having the same efficiency to ligate with different set...
Forum: Sample Prep / Library Generation 06-11-2012, 08:43 AM
Replies: 8
Views: 3,404
Posted By mikaelk
I am bit late here but I hope this might still...

I am bit late here but I hope this might still help someone.
The first question would be for me : why so much smallRNA ? or are you talking about totRNA ?
I have been using the small RNA kit for...
Forum: Sample Prep / Library Generation 06-11-2012, 08:12 AM
Replies: 2
Views: 1,753
Posted By mikaelk
In the lab we are using a directional mrna-seq...

In the lab we are using a directional mrna-seq protocol based on illumina tru-seq smallrna kits. I have not compared the two types of buffers but I have been doing polyA purifications with LiCl based...
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