transcription with dig-UTP

Andre Hamel hamel at ccu.umanitoba.ca
Tue May 18 09:20:25 EST 1993



Few things come to mind regarding in vitro transcription ...

1/	Choice of RNase inhibitor ... BEST one on market to date, without ANY
 doubt, is called InhibitACE by 5 PRIME -> 3 PRIME (500 units costs us
about $120 CDN) ... address is 5603 Arapahoe, Boulder, Colorado 80303 USA
Tel:1-800-533-5703 (anywhere in US), FAX:303-440-0835

2/	Incorporation of DIG or biotin UTP tends to be less efficient than
unlabelled UTP ... have you compared this? Have you checked your in vitro
made RNA probe on a gel? If no band(s) seen ... blot onto nylon (+ve
charged), etc ... alk. phos. detection ... what's seen then?
If/when you compare use of UTP versus DIG UTP ... how does it look on a
gel? If okay when UTP is used, then try adding DIG UTP to the UTP reaction
after about 1/2 hr into the reaction ... use varying amounts of DIG UTP
... i) "full strength", ii) 1/2, iii) 1/4, etc ... thus ... DO NOT substitute
DIG UTP totally, most of the UTP in a mix should be UNlabelled.

3/	How does the linearized plasmid look on a gel? Is there ANY
noticeable UNCUT DNA? Sometimes this affects yield ... if so, try
cleaning up the plasmid (Pro K/1% SDS dig'n ... phenol/CHCl3, etoh
ppt'n, 80% etoh washes, etc) PRIOR to overnight digestion with the
R.E. THEN repeat phenol/CHCl3 ext'ns, etoh ppt'n/washes, etc. Also, make
sure plasmid is NOT nicked ... if CsCl-EtBr purified plasmid is used, take
special care NOT to expose to UV light for any longer than needed (usually
QUICK glance with long wave UV is all that's needed for finding out where
band is ... white light usually all that's needed too since the plasmid
band will be visibly stained with EtBr. 

4/	Have you cut the plasmid on the CORRECT end of the insert?

5/	Have you considered trying AMBION's MAXIscript kit(s)? These kits
DO produce MUCH greater yields than ANY other in vitro transcription
system that I've tried (versus Promega and USB enzymes).

best luck

   ----------------------------------------------------------------------
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
Infectious & Genetic Disease                home tel.: (204) 275-1204
Mol.Biol.Lab                                FAX: (204) 945-8062
Winnipeg, Manitoba                          
CANADA
  ----------------------------------------------------------------------



In article <sticknbd-170593200703 at 129.59.16.27> sticknbd at cc.miranda.vanderbilt.edu ( sticknbd) writes:
>I am achieving low yields of riboprobe when I transcribe.
>I have a linearized, phenol/chloroform extracted insert
>in PGEM7Z, and I have used enzymes from both BMB and Promega.
>I have tried increasing the concentration of Mg, as well as
>increasing the total volume (and 10X transcription buffer of
>course), with no change in success.  Expected yield is five to
>ten micrograms with one microgram template.  I have tried it
>both with and without DNase treatment.  I use frequently-changed
>gloves, DEPC-treated water, and RNase-free glassware, as well
>as RNase inhibitor.  My yields are consistently one tenth what
>is expected or one microgram.  My insert is about 950bp, and it
>has no conspicuous areas of polyA (no tail, for example) that 
>might cause steric hindrance during transcription (only a distant
>possibility anyway).  Digoxigenin-UTP is in a cocktail supplied
>by Boehringer Mannheim (BMB), as are most other necessary components
>of the Genius labeling kit (enzymes, buffers, controls).  Transcription
>controls included in the kit work fine:  10ug yields confirmed every
>time.  I use siliconized tips and tubes.  My starting concentration of
>linearized plasmid is 1ug, but my yield is only 1ug also.  Finally, I
>need 30ul of 10ng/ul or 300ng minimum / slide (in situ), so this yield
>
>of one ug will barely do three slides.  And I prefer to use 35ul of 
>10ng/ul for hibridizations.  Any ideas why my yield is so low?



More information about the Methods mailing list