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resending of responses to PCR screening

daemon at net.bio.net daemon at net.bio.net
Mon Feb 3 17:47:20 EST 1997


This message apparently got scrambled somewhere along the line.
Here are the responces once again.  Sorry for any inconvenience.

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Several days ago I posted the following:

We wish screen transgenics for our gene of interest using PCR from a single
At leaf and have tried Wang, Qi and Cutler's 1993 method (NAR 21:4153).
We seem to be having problems even with a pair of positive control primers
that amplify fine from plasmid DNA.  Are there other reliable protocols out
there that are currently used?

This seems to be a problem experienced by others, and I had requests
to summarize responses.  Many thanks to all those who responded.

Several folks suggested that the following protocol was reliable
for both leaf and root DNA template.

"Alkali treatment for rapid preparation of plant material for
reliable PCR analysis."
Klimyuk, V.I., Carroll, B.J., Thomas, C.M., and Jones, D.G.
Plant J. 1993  3(3):493-494

Several other labs offered their own protocols, two of which follow.

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Gasser Lab:
Plant Genomic DNA Microprep

May 12, 1994, updated 8/23/96

Modified from Edwards et al., 1991  NAR 19: 1349.

This procedure is fairly rapid, we find that a person can process ~100
samples in a day.  The DNA which one obtains from this prep works well
for PCR and may be okay for for restriction digests (but we have never
used it for this).

1.  Place 400 uL of extraction buffer (200 mM Tris.Cl pH 7.5, 250 mM
NaCl, 25 mM EDTA, 0.5% SDS) into autoclaved ufuge tubes.  Shut the
cap of an eppendorf tube on a leaf to clip out a section of tissue.  Wear
gloves and minimize handling.  Rosette leaves are optimal but cauline
leaves also work.

2.  We use "disposable" polypropylene pestles that fit exactly into 1.5 ml
microfuge tubes to grind the tissue using a drill-like variable speed motor.
The tissue is ground in extraction buffer until it is well mashed and a
significant amount of chlorophyll has been released into the buffer.  We do
not dispose of the pestles, but soak them in 0.5 M HCl, rinse them and
autoclave them to them use over and over again.  (Alternatively one can
macerate the tissue in a microfuge tube with the end of a blue 1 ml
pipetman tip, but it takes much longer, and you should vortex after grinding
this way).

3.  Spin in ufuge 5 min.

4.  Transfer 300 uL of supernatant to new tube.  Try to avoid plant tissue.

5.  Add 300 =B5L 2-propanol.  Leave at RT for 2 min.  Spin in ufuge for 5
     min.

6.  Decant supernatant.

7.  Rinse with cold 70% ethanol, drain and dry pellet.

8.  Resuspend pellets in 100 uL of TE.

Notes
3.  I don't know how important this (vortexing) is but I do it anyway.

5.  The prep will still work if a small amount of plant tissue is accidental=
ly
     carried over at this step.

6.  The protocol calls for vacuum drying, but I simply invert the tubes on a
     paper towel and wait awhile.

8.  The protocol says to use 2.5 uL per PCR reaction.  I have never used
    less than this.  I have used up to 5.0 uL in PCR reactions with no
    deleterious (or beneficial) effects.
- samples should be stored frozen at -20=B0C or they will degrade over time.


***************************************************************


Arondel Lab:

1 - Place a leaf or a piece of tissue in a Kontes microtube and grind it
     with a pestle for a few seconds. (in the case of roots, or of a more
     resistant tissue, freezing the material in liquid nitrogen allows a mor=
e
    efficient disruption of the tissue).
2 - Add 400 ul of extraction buffer (200mM Tris HCl pH 7.5, 250 mM
     NaCl, 25 mM EDTA, 0.5% SDS). Grind the tissue again for 15-30 sec.
3 - Microfuge 1-2 min. Transfer 300 ul of the supernatant to a fresh tube.
4 - Add 300 ul isopropanol or 650 ul ethanol.
5 - Centrifuge 5 min. Remove the supernatant, rince the pellet with 70%
     ethanol, and dry it under vaccuum.
6 - Resuspend in 100 ul TE.

=46or small amount of tissue only, such as 5 mm of root:

7 - Add 300 ul of NaI solution (GeneClean), mix and add 2 ul of glassmilk
(GeneClean), mix again. Leave 5 min. at room temperature, and mix from
time to time.
8 - Centrifuge 1 min, and wash twice with NewWash (GeneClean).
9 - After the last wash, centrifuge again to remove any droplet of NewWash
      left.
10- Resuspend the pellet in 10 ul TE, and incubate 5 min. at 65=B0C.
11- Spin down for 3 min. and transfer the supernatant to a fresh tube.


Note: When using normal conditions of PCR, with normal oligos and
template (18 -20 mers, 50%GC), the main problem that can occur is the
presence of
junk in your DNA samples. If your PCR does not work, you should either
GeneClean your DNA or REDUCE the amount of DNA. Do not forget that
it is possible to amplify a single gene from 10 pg of Arabidopsis DNA. (Sinc=
e
you have made DNA from roughly 50 mg of tissue, you should obtain 500 ng
of DNA, which gives you a concentration of 5 ng/ul DNA - that is, a lot of
DNA).


**************************************************************

     John Shanklin
     Dept. of Biology, Bldg 463
     Brookhaven National Laboratory
     Upton, NY, 11973
     Phone:  516 344 3414
     Fax:      516 344 3407
     e-mail shanklin at bnl.gov






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