Can you elaborate your question a litle bit? We can try to help if you give more information on what you are trying to do.

I use the mmi CellCut Plus to perform the laser microdissection (http://www.molecular-machines.com/products/lasermicrodissection/mmi_cellcut_plus/overview_30388.html). During the procedure of microdissection, the fragments of the tissue are being placed on the adhesive surface of cap. I have difficulty in removing the fragments from the cap to do the DNA extraction.

I have been looking at a video of the system you use and noticed the sample indeed sticks to the cap. We use a LMD system (Leica) where the selected area is shot out and falls, due to gravity, inside the cap of a tube. The extraction buffer is placed in this cap too, either before or after the capturing. Thus when spinning the tube the sample is spun down as well. Now to translate this to your case I would recommend to add the extraction buffer in the tube, then close the lid and leave the tube up side down in order to let it extract the desired amount of time, followed by spinning it down. Another solution could be to ask the manufacturer of the Cellcut Plus system.

Thanks for your suggestions, Irene.

I've already done this way and the results were successful.

Best wishes

We use the PALM system from Carl Zeiss. While reading their protocols came across this capturing buffer which might be useful for you. By adding this buffer to the cap, you can avoid buying costly adhesive cap tubes. The procedure goes like this:

When using “unfilled” routine microfuge

tubes it is necessary to add a liquid into

the cap to facilitate the adhesion of the captured

cells.

The detergent Igepal CA-630 in the capturing

buffer allows to smear out a small amount of

liquid in the whole cap area.

Note: All kinds of aqueous solutions will run

dry with extended working time.

0.05 M EDTA pH 8.0 20 μl

1 M Tris pH 8.0 200 μl

Igepal CA-630 (SIGMA #I-3021) 50 μl

(Proteinase K )* (100 μl)

ddH2O fill up to 10 ml

*Proteinase K 20 mg/ml

Final Concentration: 20 mM Tris, 0.1 mM

EDTA, 0.5% Igepal, 1% Proteinase K

Always prepare a fresh mixture of

Capturing Buffer and Proteinase K.

Collection Procedure

1. take an autoclaved microfuge tube

2. pipette 3-15 μl of Capturing Buffer

without Proteinase K or DNase-free

water in the middle of the cap

3. put the cap/tube into the collector

check the right position of the correction

collar (see page 6)

4. perform non-contact LCM of selected cells

5. centrifuge the tube at 10000 rcf for 5 min

(Tabletop Microcentrifuge)

6. add 10-15 μl Capturing Buffer containing

Proteinase K and mix by pulse-vortexing

for 15 sec

7. incubate the tube at 56°C for 2-18h with

occasional agitation

8. centrifuge the tube at 10000 rcf for 5 min

(Tabletop Microcentrifuge)

9. final heating step at 90°C for 10 min to

inactivate Proteinase K

10. centrifuge the tube at 10000 rcf for 5 min

(Tabletop Microcentrifuge)

Note: The time necessary for complete

Proteinase K digestion depends on the

kind and the amount of collected material.

After the Proteinase K digest and the inactivation

step the routine downstream

application of choice can be continued.

If not going on immediately, store the

samples at -20 °C.

Hope it helps! Happy tissue and cell laser surgeries!

Sathya Srinivasan

Manager

RUN Microscopy Core Facility

University of Calgary

www.ucalgary.ca/runcore

Thanks, Sathya.

I'll check this possibility.

Best wishes,

Vanessa

Just a comment on the last answer from Sathya. The PALM system is completely different from the MMI CellCut: the principle of the MMI microdissection procedure is that specimen is placed on a special holder, which is covered by a membrane; the collection cap should stick on the membrane and has no direct contact with the specimen. When the cut has been completed, the samples isolated can be removed with the aid of a special adhesive film on the lid of the test tube that ensures that only the cut out parts of the specimen remain adhered to the lid. Without this special adhesive cap it is not possible to work with the MMI CellCut.

Dear Vanessa,

please check the attached publication. The authors nicely explain how to extract DNA of laser microdissected tissue isolated using the MMI CellCut system.

Do not hesitate to contact me or my colleagues for any further questions; we are happy to help!

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