single worm PCR to check transforming DNA?

Andy Vierstraete andy.vierstraete at rug.ac.be
Mon Nov 17 12:04:10 EST 1997


On 15 Nov 1997 10:38:01 -0800, qin ling <"qin ling"@MEDEW.NEMA.WAU.NL>
wrote:


>I am working on a parasitic nematode. I am now thinking transforming C
>elegans with my gene of interest tagged with gfp. This construct together
>with rol-6 will be injected into the distal gonad. My question is: if I
>could not detect any gfp expression or if I want to support the gfp
>expression, can I do a PCR on a single transformed worm with specific
>primers for my gene? Would I get a single clear signal or due to the large
>tandem array formed by introduced DNA mixture, I would  end up with a
>smear
>on my gel. I presume this probably is already a routin experiment in C.
>elegans labs.

If your primers are specific dat they only fit on 1 position in the
DNA, you should have one single band.  (If your primers are 20 or more
bases long, you will probably have a single band)

>I would be grateful if somebody can send me a single worm
>PCR
>protocol and hopefullly also answer the above mentioned naive question.

We use a protocol for PCR with nematodes of the 18S part of the gene
and it works most of the time : (from Caenorhabditis elegans Modern
Biological Analysis of an Organism by Henry Epstein and Diane Shakes)
(p 92)

1.Transfer a single worm with a platinum wire to a 2.5 ul drop of
lyses buffer in the cap of a 0.5 ml tube for thermocycling.
2. Check if the worm is in the drop of lysis buffer.
3. Close the tube and centrifuge briefly to move the drop to the
bottom of the tube
4. put the tube at -70 degrees for 10 minutes
5. Add a drop of mineral oil to the tube and heat to 60 degrees for 1
hour followd by 95 degrees for 15 minutes.  Keep one ice.
6. Add PCR mix  to the tube, mix and spin down.
7. Perform a normal PCR.

I hope it works for you, and if you want more information, let me know

Greetings,

Andy
>
>With many thanks!
>
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