PCR on intact tissue

clincoln at NATURE.BERKELEY.EDU clincoln at NATURE.BERKELEY.EDU
Sun Dec 5 12:55:36 EST 1993


Here are some of the responses I received to my question about PCR on
intact Arabidopsis tissues. Thanks to all who sent me a message.
Cindy Lincoln
PGEC
Albany, CA 
 
> Has anyone used the procedure for doing PCR on intact plant tissue
> described in Klimyuk et al, The Plant Journal (1993)3:493-494 on
> Arabidopsis tissue (such as seedlings)?  Have other procedures been
> developed for PCR on intact tissue?
 
--------------------------------------------------------------------------------
I have been using the procedure described in Klimyuk et al on sprout
tissue.

Basically I sprout the plants on wet filter paper and clip them off the
paper and into the NaOH as soon as they have green tops and are long enough
to reasonably get a hold of. I've been putting about 4-10 sprouts in each
tube, but it has worked with as few a s 2 and as many as 15.

We've been using this technique to do SSLP screening.It has allowed us to
go from seed to scoring the sprouts for RFLPs in about 5 days.I have been
able to score the amplified product with about an 87% success rate. I
suspect that the variability has to do with differences between the two
primers I'm using and the higher concentration of contaminants from the
filter paper, the seed coat, and the fungus and mold which grows on the old
sprouts (I haven't been sterilizing the seed).

Hope this info is of some use to you.

-Airlie Sattler


Cindy,
        Regarding the PCR of intact tissue. It is great! I routinely
use it on cauline leaves of Arabidopsis thaliana. One sample was
a small piece of stem which failed to break up with a micropipette tip
so I added one microlitre of the solution and got a signal. Have also
used a very dried up dead leaf and obtained a signal. No worries.

Cameron Johnson
Genetics and Developmental Biology
Monash University
Melbourne, Australia


Hi. I have used the Plant J. procedure, and it usually works. I have yet to
determine if my problems are primer-specific or PCR machine specific, or
both.
Ricardo Azpiroz
Plant Sciences
U. Arizona.


 We used the procedure of Berthomieu and Meyer (plant. mol
Biol 17: 555-557 {1991}) successfully on leaf material
from sterile plants.
 However, I would stress using sterile plant 
material, our first attempts on soil grown plants yielded a 
homolog to the gene we were interested in, but this did 
not cross hybridise on Arabidopsis Southern blots.

Hope this is of some help

Neil Butt
Bilogical Sciences
University of Sussex
Brighton, BN1 9QG.
UK


Dear Cindy,

I have tried the Klimyuk method using individual flowers, half an
inflorescence, and a silique.  I essentially followed the protocol as
published, and I had very good success with individual flowers, and
progressively less success with the more material that I used.  I had NO
success with siliques, even rather young ones.  Good luck!  Leslie Sieburth
(Meyerowitz Lab)

Klimyuk's protocol works beautifully on Arabidopsis seedlings and is simplest
I know of.
Dima Belostotsky
dimtry at bscr.uga.edu


I have used intact tissue for PCR without any problem. I simply cut off a
tiny little bit of material and used it directly in a PCR reaction. All
material, even dried siliques worked fine, except for intact seeds. I
believe that the cutting opens enough clls so that PCR is started from the
damged cells. When I used roots, I saw a strong correlation between amount
of material and successful amplification. The less material the better.
What I routinely did was punching little leaf discs out of leaves using 
 yellow pipettip. This tiny piece was then eluted into the PCR solution.
Sucking the PCR solution up and down with the pipet without even displacing
the leafdisc produced excellent results. Rigorous negative controls were
included and I never had any fals positives. This methods may be very
useful for screening lots of individual seedlings, since you don't have to
do anything to the seedlings except for punching a hole into the leaf.

Roger Aeschbacher   Dept. of Biology New York University




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