Guidelines for sample handling (F-130)

Introduction

To obtain small and uniform samples, we recommend using the Harris tools provided in the kit for sampling. The Harris Uni-Core may be disposed of after use or cleaned and reused up to 500 times, depending on the thickness and firmness of the sample material. If the puncher is to be reused, it is very important to clean the cutting edge properly to prevent cross-contamination between samples (see Step 2 for cleaning instructions). The Harris Cutting Mat provides the best possible cutting surface for Harris Uni-Core. It is made of inert self-healing material and has two cutting surfaces. The cutting mat can be reused several hundred times, but it should be cleaned between each sample to prevent cross-contamination (see Step 2). The Harris tools are also available separately from Finnzymes.

Protocol

1. The use of Harris Uni-Core
   
   The cutting edge of Harris Uni-Core is very sharp. Therefore, special attention is required when working with this puncher.
   Please follow the instructions below.
   
   1. Place the sample on the Harris Cutting Mat.
   2. Remove the protective cap from the cutting edge of the Harris Uni-Core by twisting it gently or flipping it off using your thumb.
   3. Holding the puncher firmly, push the cutting edge downward into the sample and rotate the puncher in opposite directions until it cuts through the sample. Only very gentle downward pressure is required. Do not press the plunger while cutting.
   4. Lift the puncher away from the sample and press the plunger to eject the punch disc into a PCR reaction. Make sure that the sample drops into the PCR solution and does not stick to the tube walls.
   5. Clean both the Harris Uni-Core and the Cutting Mat after every sample as described in Step 2.
   A video showing the sampling and cleaning procedure is available at www.finnzymes.com/directpcr.
2. Cleaning the sampling tools 
   
   To prevent cross-contamination between samples, it is important to clean the cutting edge of the Harris Uni-Core between each sample. Dip the cutting edge into 2 % sodium hypochlorite* (NaClO) solution and press the plunger up and down a few times. After rinsing, wipe the tip with a clean paper towel. The Cutting Mat should also be rinsed with 2 % sodium hypochlorite solution after each sampling. 
   
   It is recommended to include a negative control without DNA template in all assays. To monitor cross-contamination, the cleaned puncher can be dipped into the negative control sample (see Step 3). For more information, refer to the application protocol about avoiding cross-contamination when using the Harris Uni-Core (available at www.finnzymes.com/directpcr).
3. Plant leaves 
   
   Direct protocol
   Take a sample from the plant leaf using the 0.50 mm Harris Uni- Core puncher supported by the Cutting Mat. Place the punch disc directly into the PCR reaction (20−50 μl in volume). It is recommended to eject the disc into a liquid, rather than onto the wall of an empty tube. Make sure that you see the sample disc in the solution. We recommend using young leaves. Fresh plant material is usually the best choice, even though plant material stored at +4°C, frozen or on commercially available cards such as Whatman 903 and FTA cards can also be used (see Step 5). For amplifying long fragments or difficult samples using the direct protocol, a 0.35 mm punch disc may give more robust results. A Harris Uni-Core 0.35 mm is available separately (F-180S/L). 
   
   Dilution protocol
   As with the direct protocol, young leaves are recommended. Take one small leaf or a piece of leaf (e.g. a punch approximately 2 mm in diameter) and place it in 20 μl of Dilution Buffer. Crush the leaf sample with a 100 μl pipette tip by pressing it briefly against the tube wall. If larger amount of leaf tissue is used (do not exceed 1 mg), increase the volume of the Dilution Buffer to 50 μl. After crushing the leaf, the solution should be greenish in colour. Spin the plant material down, and use 0.5 μl of the supernatant as a template for a 20 μl PCR reaction. The required volume of the supernatant may vary depending on the plant material used and the volume used for the dilution.
4. Plant seeds 
   
   Direct protocol
   Using a clean scalpel, remove the seed coat and cut a small sample of the seed (approximately the size of this dot •) supported by the Cutting Mat. Place the sample directly into the PCR reaction (20−50 μl in volume). Note that it is recommended to use dehulled seeds. For very small seeds (such as Arabidopsis), use 1−2 whole seeds and place them directly into the PCR reaction. 
   
   Dilution protocol
   Cut a small sample of the dehulled seed by using a scalpel (approximately the size of this dot •) and place it directly into 20 μl of Dilution Buffer. Briefly vortex the tube and incubate at room temperature for 3 min. Make sure that the seed sample is covered with Dilution Buffer. Spin briefly and use 0.5 μl of the supernatant as a template for a 20 μl PCR reaction.
5. Plant material stored on commercially available storage cards, e.g. Whatman 903® and FTA® Cards 
   
   Direct protocol
   Use the 0.50 mm Harris Uni-Core to cut a disc from the sample in the storage card. Place the punch disc directly into a 50 μl PCR reaction. For amplifying long fragments or difficult samples, a 0.35 mm punch disc may give more robust results. A Harris Uni-Core 0.35 mm is available separately (F-180S/L).