PEG/NaCl ppt. of PCR products

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Mon Dec 22 14:33:28 EST 1997


At 19:22 12/11/97 +0000, C.S. Wilding wrote:
>Is there a lower cut-off point for PEG/NaCl 
>precipitation of PCR products prior to sequencing (as 
>referred to in Current Protocols). I usually use it for 
>600-1000bp products and it is OK but failed in an effort 
>on 120-180bp products. Is the method unsuitable in this 
>instance?

Craig:

Sorry for the delay in replying to your post...

What number of bp are precipitated depends upon the composition of the PEG
solution.  Two the initial references describing the use of PEG to
precipitate DNA was Lis (1980) Meth. Enzy. 65:437 and  Paithankar & Prasad
(1991) NAR 19:1346.  PEG shuts down virtually all enzymes except for T4-DNA
ligase  (Teraoka (1987) J. Biochem. 101:225-231).  Fortunately, one of the
many enzymes that PEG inhibits is Taq (Barnes, (1992) Gene 112:29-35).  Up
until recently, most papers employed using anywhere from 10-13% PEG for
"general" precipitation of DNA and almost always used  a solution
containing 40% PEG (w/v) and1.5 M NaCl within.

Then Nicoletti,VG et al published a very slick mini prep protocol in 1993
BioTechniques 14:532-536, wherein they claimed that PEG with NaCl
inconsistently precipitated DNA below 500 bp and when below a critical
threshold of 1 ug/ml.  However, when they substituted 30mM MgCl2, then
precipitation was consistent down to about 120 bp using a final of 13% PEG.
 [Personal note, this is one of the few homegrown mini preps that is
moderately quick, cheap, and will yield DNA that can be sequenced on an
automated DNA sequencer and whose DNA is "transfectable".]

With no year printed on the single sheet article, I came across a "Focus"
article wherein the author (a Gibco res. scientist)  kept the MgCl2
concentration the same, 30mM, but varied the PEG concentration.  At 10% PEG
all bands down to about 120 were precipitated.  With 8.3% PEG, not much
below 200bp was recovered, with 6.7% I'd guesstimate not much below 450-550
was recovered and at 5%, ZILCH...  As we know, the amount of PEG to enhance
ligations is roughly 4-4.5% so failure to precipitate DNA using 5% was no
surprise.

I've used PEG for quite a number of purposes, namely trough elution where
the recovered yield can be quite high (see Zhen and Swank (1993)
BioTechniques 14(6):894-898.   However, one must be very religious about
washing it out of the DNA preparation if it is to be used for anything
other than ligations.  Superstitiously, I wash the DNA pellet no less than
5 times with vortexing using 70% EtOH and even with that sometimes I'll get
a probe that won't label as well as one prepared through PEG-less methods
(trough, freeze squeeze, etc.)

I hope this helps,
David

=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu"
 http://www.uth.tmc.edu/~dhavilan 
 Voice: 713.500.2413  FAX: 713.500.2424
"A conclusion is simply the place where you got tired of thinking."
=============================



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