Three protocol PDFs attached that I stumbled upon on the web, I thought some of you may be interested.
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454 Library / Emulsion / Instrument Protocols (October 2008)
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cDNA from Evrogen with Ti
Has anybody had any experience with Ti sequencing Evrogen Trimmer Direct cDNA? For a 2 region Ti, we got 67 Mb and only 20% pass. The support people feel it is a problem in the emulsion PCR. Any help would be much appreciated. We've sequenced ~30-40 similar samples with FLX and no problems (over 100 Mb, ~440,000 reads per run).
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Hello, I´m a new member.
Originally posted by ECO View PostThree protocol PDFs attached that I stumbled upon on the web, I thought some of you may be interested.
Thank you for the three protocol pdf!Last edited by futuro_cortina; 03-09-2009, 11:21 AM.
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PTS Wisdom?
Hi
We are currently working on de novo RNA-seq and re-sequencing in a pre-genome species (onion) and are interested in PTS or a similar generic barcoding approach to enable us to do population genetic studies.
If anyone can give me any opinion or advice concerning this approach I'd be really grateful. To date we are just working with barcoded primers
Regards
John
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Hi
We are trying to work out cDNA sequencing using 454, but Titanium did not work, and Standard was severely affected by polyA tail despite the nice profile of the sstDNA library.
We used Dynabeads for mRNA purification, and Stratagen Just cDNA kit for ds cDNA synthesis (1st strand primed by none-anchored oligo dT from the kit). Our samples tend to have long polyA tail, and we found 454 sequencing cannot tolerate this.
We are trying anchored dT primer now, but would appreciate any suggestion, such as choice of kit, 1st strand priming method (dT vs random primer?), or any tips in improving library constrcution for greater chance of success in sequencing?
Thank you.
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re Poly A etc
We have had very nice results from Ti sequencing of normalised cDNA prepared using Evrogen Mint + Trimmer kits, using a modified 3' Primer XXXXXXXXXXX(T)4G(T)9C(T)10VN as described by Beldade et al.
The information posted at http://www.cees.uio.no/research/faci...equencing.html was very useful.
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