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Yeast genes for arraying

Francis Ouellette francis at cmmt.ubc.ca
Thu Dec 9 10:28:20 EST 1999


This is a cross-post from another non-bionet newsgroup, but I think
useful for the yeast community to know about.

cheers,

f.

--
| B.F. Francis Ouellette                      Tel: (604) 875-3815 | 
| Director, Bioinformatics Core Facility      Fax: (425) 740-6978 | 
| CMMT, UBC, Canada                        http://www.cmmt.ubc.ca | 
| francis at cmmt.ubc.ca                http://www.bioinformatics.ca |



> Date: Thu, 9 Dec 1999 09:55:57 -0500
> From: Michael Lichten <lichten at HELIX.NIH.GOV>
> Reply-To: To share info regarding GeneChips tech. and Microarrays in           
>      general <GENE-ARRAYS at ITSSRV1.UCSF.EDU>
> To: GENE-ARRAYS at ITSSRV1.UCSF.EDU
> Subject: Re: Yeast genes for arraying

>Following previous discussions concerning the Research Genetics Unigene
>clones, I was wondering weather anybody has experience with the Research
>Genetics yeast products for arraying (either the amplified Yeast ORFs or
>the Yeast GenePairs) .
>Any inputs will be appreciated.
>
We have used individual genepairs from ResGen with good success to
amplify all ORFs on a single yeast chromosome; failure rate once PCR
is optimized (NOT ResGen's protocol) is about 5%.  Secondary
amplification with the universal priming sequences supplied on the
GenePairs was also successful, yields are superb and frequently
rescue primary PCRs that appear to lack a product.  About 1/2 of the
"failures" are rescued this way; the remaining failures split about
evenly between ResGen mistakes (i.e. were corrected when we asked
them to resynthesize the primers) and likely sequence problems (i.e.
we had to use different primers more internal to the ORF).  We did
not try to amplify ORFs > 3 kb, but designed our own primers to split
them into more managable bits.

We have had much more trouble with the amplified ORFs; they were all
supplied at less than the advertised concentration, and 4/4 could not
be reliably amplified using the universal primers whereas our own
primary products (should be identical) could be done so easily.

One further note; the yeast DNA that ResGen supplies is degraded and
should not be used.

I will be glad to supply our modified PCR protocol upon request.
----------------------------------------------------------------------------
Michael Lichten                         phone:  301-496-9760
LB/DBS/NCI                              fax:    301-402-3095
Building 37 Room 4C03                   email:  lichten at helix.nih.gov
37 Convent Dr. MSC 4255
Bethesda, MD 20892-4255





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