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Forum: Sample Prep / Library Generation 10-13-2017, 07:16 AM
Replies: 8
Tn5
Views: 2,139
Posted By Olaf Blue
EZ-Tn5 from Epicentre has been transferred for...

EZ-Tn5 from Epicentre has been transferred for sales and support to Lucigen:

http://www.lucigen.com/EZ-Tn5-Transposase/

Olaf
Forum: Illumina/Solexa 09-07-2017, 05:25 AM
Replies: 17
Views: 2,041
Posted By Olaf Blue
Perhaps Illumina/Epicentre's ScriptSeq RNA-Seq? ...

Perhaps Illumina/Epicentre's ScriptSeq RNA-Seq?
...
Forum: General 08-10-2017, 09:57 AM
Replies: 1
Views: 1,125
Posted By Olaf Blue
The pCC1BAC vectors are all 8,128 bases no matter...

The pCC1BAC vectors are all 8,128 bases no matter the linearizing nuclease:

http://www.epibio.com/tech-support/dna-sequences/pcc1bac_hind

Otherwise digest the clone with the enzyme NotI as it...
Forum: Illumina/Solexa 06-27-2017, 04:55 AM
Replies: 4
Views: 738
Posted By Olaf Blue
The stand-alone Ribo-Zero kits are to be used...

The stand-alone Ribo-Zero kits are to be used with 100 ng up to 5 ug of total RNA. In the past, Epicentre/Illumina's Scriptseq RNA-Seq kits with Ribo-Zero have been used for rRNA depletion and...
Forum: Sample Prep / Library Generation 05-25-2017, 09:15 AM
Replies: 10
Views: 5,310
Posted By Olaf Blue
I typically use 80% ethanol and two washes @ 175...

I typically use 80% ethanol and two washes @ 175 ul each. Allow to dry for 5 minutes - elute with Low EDTA TE or "RSB" (Resuspension buffer). Water should be fine, too. Make sure you mix the beads...
Forum: Illumina/Solexa 04-13-2017, 10:36 AM
Replies: 19
Views: 6,033
Posted By Olaf Blue
Treatment of PCR products with Exo...

Treatment of PCR products with Exo I/EXOSAP/Antarctic Phosphatase for primer removal is an old trick and works very well.
Forum: Sample Prep / Library Generation 03-28-2017, 04:57 AM
Replies: 11
Views: 4,467
Posted By Olaf Blue
Have you tried depleting the rRNA using Ribo-Zero...

Have you tried depleting the rRNA using Ribo-Zero or RNAse H methods and seeing if or how many of the small RNAs remain or are eliminated using Bioanalyzer?
Forum: Sample Prep / Library Generation 02-08-2017, 10:03 AM
Replies: 8
Views: 1,749
Posted By Olaf Blue
OK. PVP shouldn't be necessary when extracting...

OK. PVP shouldn't be necessary when extracting DNA from bacteria.
Forum: Sample Prep / Library Generation 02-08-2017, 05:47 AM
Replies: 8
Views: 1,749
Posted By Olaf Blue
Are you using Polyvinyl pyrollidone in any of the...

Are you using Polyvinyl pyrollidone in any of the cleanup steps? Which plant/sample type are you extracting?
Forum: Sample Prep / Library Generation 01-31-2017, 06:44 AM
Replies: 2
Views: 1,399
Posted By Olaf Blue
The ScriptSeq user guide provides the complete...

The ScriptSeq user guide provides the complete sequences of the reverse/index primers. If a user so desires, they can order their own custom primers from any good oligo/primer manufacturer and simply...
Forum: Sample Prep / Library Generation 08-01-2016, 05:40 AM
Replies: 1
Views: 1,309
Posted By Olaf Blue
The main difference is the specificity of the...

The main difference is the specificity of the rRNA removal solutions in the kits.

Epidemiology = Human/Mouse/Rat plus Gram negative and Gram positive rRNA capture probes
Bacteria = Gram Negative...
Forum: Sample Prep / Library Generation 02-19-2016, 07:37 AM
Replies: 8
Tn5
Views: 2,139
Posted By Olaf Blue
Happy to help - this is just the bare enzyme and...

Happy to help - this is just the bare enzyme and users have to load any oligos themselves. Here's the product page link:
...
Forum: Sample Prep / Library Generation 02-19-2016, 06:13 AM
Replies: 8
Tn5
Views: 2,139
Posted By Olaf Blue
Epicentre's EZ-Tn5 Transposase; TNP92110, which...

Epicentre's EZ-Tn5 Transposase; TNP92110, which is a special order item.
Forum: Sample Prep / Library Generation 01-04-2016, 06:05 AM
Replies: 6
Views: 1,546
Posted By Olaf Blue
Grape leaves (Vinifera) typically have issues...

Grape leaves (Vinifera) typically have issues with complex carbohydrates and polyphenols in the tissues. Possible that these are issues for you? Sometimes, using 1% polyvinylpyrrolidone in the RNA...
Forum: Illumina/Solexa 09-02-2015, 09:36 AM
Replies: 16
Views: 11,848
Posted By Olaf Blue
We use these to clean up gel purifications: ...

We use these to clean up gel purifications:

http://www.emdmillipore.com/US/en/product/,MM_NF-UFC30GVNB
Forum: Sample Prep / Library Generation 08-31-2015, 08:25 AM
Replies: 6
Views: 909
Posted By Olaf Blue
Best thing to do is two-fold: 1) Check the RNA...

Best thing to do is two-fold: 1) Check the RNA post-Ribo-Zero on a Bioanalyzer (if the total RNA was of high quality to start with, you should see disappearance of the two major rRNA peaks (16/23s or...
Forum: Sample Prep / Library Generation 08-31-2015, 06:14 AM
Replies: 6
Views: 909
Posted By Olaf Blue
The Illumina user guide reports 8% recovery; from...

The Illumina user guide reports 8% recovery; from 5 g of RNA, up to 400 ng of non-ribosomal RNA - to include tRNAs, non-coding RNAs and mRNA (mRNA give the lowest of the recovered RNA in terms of...
Forum: Sample Prep / Library Generation 08-28-2015, 11:52 AM
Replies: 6
Views: 909
Posted By Olaf Blue
Hello- First, Nanodrop is not sensitive...

Hello-

First, Nanodrop is not sensitive enough to be able to quantify output from the rRNA depletion steps; we recommend either a Qubit with Ribo-Green and a Bioanalyzer using a Pico chip.
...
Forum: Sample Prep / Library Generation 04-15-2015, 07:08 AM
Replies: 4
Views: 1,421
Posted By Olaf Blue
Hi Exo- The modifications look ok and are...

Hi Exo-

The modifications look ok and are not that much of a deviation from the protocol. They should be fine.

A couple of other things - the StarScript RT used in ScriptSeq library prep is an...
Forum: Sample Prep / Library Generation 09-19-2014, 08:06 AM
Replies: 7
Views: 1,146
Posted By Olaf Blue
Not certain if this wll help but Jay Shendure's...

Not certain if this wll help but Jay Shendure's group performed a high throughput deep sequencing project on a Fosmid library:

http://www.nature.com/nbt/journal/v29/n1/abs/nbt.1740.html

Olaf
Forum: Bioinformatics 08-19-2014, 10:28 AM
Replies: 5
Views: 1,947
Posted By Olaf Blue
For ScriptSeq libraries, use –fr secondstrand. ...

For ScriptSeq libraries, use –fr secondstrand. -fr secondstrand means that the strand being synthesized on the sequencer is the sense strand for Read 1.

Olaf
Forum: Sample Prep / Library Generation 07-18-2014, 09:29 AM
Replies: 1
Views: 921
Posted By Olaf Blue
That should work. Epicentre includes Exonuclease...

That should work. Epicentre includes Exonuclease I in their kit but it's only referred to for use in removing excess PCR primers in FFPE applications. I was told it was holdover from the previous...
Forum: Sample Prep / Library Generation 07-11-2014, 06:11 AM
Replies: 3
Views: 2,023
Posted By Olaf Blue
The Nextera technology is unique. If you have PCR...

The Nextera technology is unique. If you have PCR products that have ligated adapters/indexes, a tagmentation reaction will fragment the products and depending on the length of the PCR products, may...
Forum: Sample Prep / Library Generation 07-11-2014, 05:45 AM
Replies: 3
Views: 2,023
Posted By Olaf Blue
Hello Ahmed_BH, Probably not. The...

Hello Ahmed_BH,

Probably not. The Transposase in the Nextera reactions has a dual purpose, to insert the oligos into the DNA and to cut the DNA. Insertion of the Transposome results in a 9-base...
Forum: Sample Prep / Library Generation 03-13-2014, 01:08 PM
Replies: 5
Views: 4,820
Posted By Olaf Blue
Hi, Drosophila is not the best substrate...

Hi,

Drosophila is not the best substrate for Ribo-Zero, but it does work (see attached data). Not all rRNA species are readily removed, but if you do a Google Scholar search under "Ribo-Zero" and...
Showing results 1 to 25 of 58

 


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