UNBELIEVABLE------=in_/\_vitro=------ T R A N S C R I P T I O N

Josmar Langner josmarlangner at yahoo.de
Tue May 4 09:13:25 EST 2004


The precipitate is indeed pyrophosphate. No idea whether Mg-ions are
trapped (no chemist). If you see it your reaction usually worked well.
Sometimes your transcription yield inceases if you get rid of it by
adding pyrophosphatase right from the start.
Josmar

alion85 at hotmail.com (redeamer) wrote in message news:<a86fd891.0405032107.3a99c514 at posting.google.com>...
> Hi guys!
> 
> Last monday I desperately needed some RNA for my RdRp assays, and of
> course was forced to make in vitro transcription: linearized template
> for some 4-5 hours (was a bit lazy), purified it using a spin column
> (was again lazy, used an eppendorf perfect prep kit [nice thing]).
> Generally i am doing my transcription for 3 hours in some 30-40
> microliters, I use T7 polymerase. However, that day was exceptionally
> lazyas ordinarily i have put 1U of polymerase per microliter of magic
> mixture ([rNTPs] were about 1 mM, DNA was some 3,5 micro-grams ),
> incubated for 2h  @ 37, and then i was again lazy and decided to add
> some more of polymerase (+ 1U/microliter), and incubated again for 2
> hours...
> 
> When i removed my tubes from the heater i saw a discrete precipitate
> at the bottom, I centrifuged it a bit, removed the super, added DNase
> I, extracted with acidic phenol (btw for those who doesnt no hau to
> make an acidic phenol in 30 sec: 1:1:0.2:0.04 =
> phenol:chloroform:10%SDS:0.5M EDTA), precipitated with
> isopropanol:ammonium acetate and washed with EtOH....
> 
> well the thing i saw at my gel picture was nice i had some 3 microgram
> per microliter RNA concentration with a total of 60 microgram from 3.5
> microgram DNA input...
> 
> I suppose that the precipitate was the Mg2+ surrounded by a cage of
> pyrophosphates, what indicated the throughout extensive exhausted
> utilization of my rNTPs...
> 
> Can anyone share some light on this interesting phenomenon:)))) May be
> I am mistaken
> 
> 
> -----> Drew



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