PCR Trouble

Andre Hamel hamel at cc.umanitoba.ca
Sat Jun 4 11:13:30 EST 1994



Assuming the genomic DNA doesn't come from whole blood, but instead from cell
culture, try simply treating the DNA sol'n in 0.1% SDS, with Proteinase K
at 55*C for about 30 min, followed by phenol/CHCl3 ext'ns x2, CHCl3 ext'n
(1 or 2) then add salt (0.3 M NaAc), then 2x vol EtOH, etc.

If DNA is from whole blood or bloodddy tissues, I suspect that blood borne
inhibitors of PCR may account for problems ... there's many articles in
bionet archives citing good methods invovling use of charged resins
(chelex and like) ... that failing, I routinely extract DNA (&/or RNA)
from small samples of whole blood and bloody tissues using modification of
protocols described by Macfarlane & Dahle (nature, Mar, 1993) ... involves
their patented cationic detergent called Catrimox-14 (tetra decyl
trimethyl ammonium oxalate), which forms ppt with anionis such as RNA &
DNA ... then one simply selectively extracts RNA or DNA (acidic GITC [4 M
with 0.2 M NaAc, pH 4.0) and acidic phenol (dH2O sat'd) & CHCl3 ext'ns of
Catrimox pellet to yield RNA, or alkaline, pH 8 to 9, GITC (4 M with 0.2 M
NaAc, pH 8 to 9, adjusted dropwise with 1 N NaOH) and alk phenol (pH 8 to
9 Tris sat'd) & CHCl3 ext'ns for DNA.

For blood (citrated works best, though EDTA blood pellet can be washed in
1x SSC (0.3 M NaCl/30 mM Nacitrate, pH 7) ... added to 10x volume of
Catrimox ... vortexed ... sit rm temp 10 min ... ufuge 5 min ... drain,
quick spin, suck off remaining sup ... dissolve pellet in desired GITC
(acidic for RNA, alk for DNA) ... phenol & CHCl3 ext'n as needed, etc ...
I've ALWAYS found this to PCR (RNA-PCR and DNA-PCR) exceptionally well ...
heme, etc is removed dur to SELCTIVE ppt'n of nucleic acids (& SOME other
polyanionics) during Catrimox lysis step.

For tissues, simply first homogenize in desired volume of PBS (5x vol), quick
spin, add sup to 5 to 10x vol Catrimox, vortex, etc as above.

Costs about $1 US per mL, for us, it's a bargain considering we
routinely survey at least 100 specimens daily (small specimens tested,
that is, a few drops of blood, or few drops of tissue homogenates obtained
from 5 grams tissue) ... yields are between 1 abd 20 ug DNA and 10 and 100
ug RNA (depending on type of & state of specimen).

If any interest, I'l'l post MORE details (I have a several page protocol
with references, but I felt was too long to post here right now).

all best,

Andre


In article <Cqu2Jr.MM5 at news.cis.umn.edu> wilden at lenti.med.umn.edu (Scott Wildenberg (DGM-IHG)) writes:
>I have been trying to amplify a region of human genomic DNA.  I have 
>amplified it successfully in the past, but, under the same conditions, 
>some new samples are not working.  I have done a phenol/CHCl3 extraction 
>and ethanol precipitation to clean them up, run the reactions with MgCl2 
>concentrations ranging from 0.5 mM to 4.0 mM, and tried increasing the 
>amount of DNA.  I got a very faint band when I increased the amount of 
>DNA to 400ng (50 ul rxn).  I figure it must be something about the 
>samples because I have run positive controls, and they always look great.
>Does anyone have any advice on what I can do?
>
>
>Thanks,
>
>Scott C. Wildenberg
>
>Division of Genetics and Metabolism   	Voice (612) 625-5628
>University of Minnesota               	Fax (612) 624-6645
>Box 485 UMHC                            Email wilden at gene.med.umn.edu
>Harvard Street at East River Road     
>Minneapolis, MN  55455                   
>





More information about the Methods mailing list