It is recommended to handle samples in the following manner:

• Use a clean and dry sample tube rack.
• Put on a fresh pair of gloves.
• Check that the sample tube lid is closed firmly.
• Then mix the sample by swirling the tube strongly.
• Wipe the outside of the sample tube using an ethanol wipe.
• Guide the sample down into the bottom of the tube by gently tapping the tube on the bench.
• Change gloves before handling the next sample tube.
• Leave samples to sit for 15 min before testing to allow the plant material to settle to the bottom.

Follow the steps in the same order to ensure the best protection of the testing integrity.

• If you have long hair, tie it back to keep it away from your face and samples and equipment.
• Wear appropriate protective clothing such as a clean long-sleeved shirt or a clean dedicated coat.
• We also encourage the use of close-fitting safety glasses to protect your eyes.
• Then put on a pair of gloves.
• Once you have put on your gloves, you should avoid touching unnecessary items. For example, don't touch your skin, hair, the chair, the ground, or door handles with your gloves. If anything drops on the ground, leave it there until the end of the testing.
• Clean gloves with an ethanol wipe and dispose of the ethanol wipe in a plastic bag. You could use an empty bread bag for this purpose.
• Wipe the bench area with an ethanol wipe before the testing.
• Wipe down the tube racks, the outside surface of the tip box, and the micropipette with ethanol wipes.
• Perform the testing following the test instructions. Refer to the accompanying video and written instructions for the testing.
• All used or contaminated tips should be disposed of into a disposal bottle with a lid that can be closed before disposing of the bottle into general rubbish bins. You could use an empty milk bottle for this purpose.
• At the end of the testing, dispose of the reagent tubes into the disposal bottle and close the lid of the bottle.
• You can also dispose of the sample tubes into the same bottle once you have your results and no longer need the samples.
• After testing is completed, wipe down the equipment and bench area again with ethanol wipes before putting the equipment away.
• Dispose of your gloves in the plastic bag. Seal the bag either by tying a knot in the bag or using a rubber band before disposing into general rubbish bins.

Chemical Safety

There are no known hazards identified with the chemicals used in this kit. However, individuals can have varying sensitivities to different reagents, and while only very small quantities are supplied, care should always be taken to avoid exposure of skin and eyes to these chemicals.

When storing the kit reagents, consider the location and manner of storage to ensure they are kept out of reach of children or other individuals who don’t know how to handle them. If contact is made by kit reagents with skin or eyes, flush thoroughly with water.

Next, use the micropipette to measure 20?L of liquid from a sample tube into a reagent tube.

• Place the sample tube into the sample rack. Place the reagent tube into the reagent rack.
• Remove the lid of a disposable bottle. You could use an empty milk bottle for this purpose.
• Loosen the lid of the sample tube.
• Hold the micropipette in one hand.
• Open the lid of the tip box with the other hand. Do not touch the tips.
• Gently insert the bottom end of the micropipette into the top of a tip. Tap gently to check the tip stays on.
• Lift the pipette away with the tip on the end and close the tip box.
• Using your free hand, place the sample tube lid upside down on the bench. Now, pick up the open sample tube without touching the top and inner edge of the sample tube.
• Press the white button on the top of the micropipette down to the first stop and hold.
• While holding at the first stop, place the tip into the top layer of the liquid in the sample tube.
• Slowly release the white button upwards to draw up the liquid.
• Lift the tip out of the tube.
• Place the tube back into the rack and set the lid on top.
• Pick up the reagent tube and carefully open it using your thumb to press upwards on the lid. Avoid touching any part of the lid that goes inside the tube.
• Place the tip inside the reagent tube and just into the top of the reagent liquid.
• Press the white button slowly to the first stop to eject the measured liquid.
• Mix the contents by slowly releasing and slowly pressing down the white button 4x.
• At the last downward button press to the first stop, hold, and then lift the tip just clear of the liquid.
• Touch the tip to the inner wall of the tube just above the liquid and press the button further through to the second stop and hold to eject all the remaining liquid onto the tube wall.
• Move the tip off the wall of the tube whilst holding the button at the second stop.
• Slowly release the white button and lift the tip clear of the tube.
• Close the tube lid without touching any internal parts and place the tube back into the reagent rack.
• Discard the tip into the disposal bottle by holding the tip just inside the neck of the disposal bottle and pressing the black tip ejector. The tip should drop off into the bottle.
• Place the micropipette back on the stand.

Always use a new tip between samples and for each reagent.

Be aware of where the tip touches. If you bump a rack, the bench, or the outside of a tube, then you must discard that tip.

If a used tip falls onto the bench, leave it there until the end of the testing.

Do not leave tips on the end of the micropipette when on the stand, as they will not stay clean, or they might get used twice, creating contamination across samples and/or tests.

• Prepare yourself and the testing area as outlined in the Hygiene and Safety section.
• Each test strip can test up to 6 sugarcane xylem samples.
• Prepare sample tubes as per the Handling samples section. Mix each sugarcane xylem sample tube and then allow them to stand for 15 min in the middle positions of the front row in the Sample rack (see the Sample rack diagram below). The positions at either end (red and green) is where the control sample tubes will be sitting later.

• Turn on the heat block to 67°C.
  • The heat block will have been pre-set before delivery. If not, follow the instructions in the booklet provided with the heat block.
• Complete the sample order table on the results sheet. Spare results sheets can be downloaded here
  Results Sheet
• Remove all reagent bags from the fridge and the freezer.
  • 1x bag of (two) control tubes (from fridge)
  • 1x bag of (eight) Reagent 1 tubes (from fridge)
  • 1x bag of (eight) Reagent 2 tubes (from fridge)
  • 1x bag of (eight) Test reaction tubes (from freezer)
• Open the Control sample tubes and place them into the Sample rack.
• Check that the control and sample vials are in the same order as the table on the results sheet.
• Open the bags for Reagents 1 and 2 and place them into the Reagent rack (see the suggested positioning in the Reagent rack diagram below).

• Remove one packet containing 1 strip of 8 tubes of freeze-dried reaction mix from the freezer. Allow the pack to come up to room temperature while processing samples before opening.
• Samples should be processed through the buffers fairly promptly, with the time in buffer 1 no longer than 5 min. They can be processed either individually or as a group through the buffers.
• Working from right to left:
  • Add 20?L of the sample into Buffer 1
  • Pipette gently up and down 4x to mix.
  • Close the lid and mix by gently flicking the bottom of the tube with a finger.
• Again, working from right to left:
  • Add 20?L of the “sample/Buffer 1 mix” into Buffer 2
  • Pipette gently up and down 4x to mix.
  • Close the lid and mix by gently flicking the bottom of the tube with a finger.
• Check that the temperature of the heat block has reached 67°C.
• Open the packet of freeze-dried reaction mix tubes and place the tubes into the Test rack (see the Test rack diagram below). Carefully close any lids that may have popped open without touching the internal parts.

• Again, working from right to left:
  • Add 20?L of the “sample/Buffer 2 mix” to the reaction tubes.
  • Flick gently to mix.
• Leave tubes to sit for 3 min, flicking gently every min
• Check that all the lids are firmly closed.
• Up-end the tube strip sharply 5x to mix and also to catch any pink reagent that might be sitting further up the tubes.
• Ensure all liquid has returned to the bottom of the tubes by flicking the strip downwards.
• Place the tubes into the heat block at 67°C.
• Start a timer for 40 minutes.
• At the end of the 40-minute incubation period, open the lid of the heat block carefully and check that all tube lids are firmly closed.

It is very important that these incubated tubes are not opened, as the levels of DNA will have increased substantially and can be a source of contamination to the surrounding area and equipment, affecting future test results. If this issue occurs, follow the decontamination protocol below.

• Remove the strip of tubes from the heat block, and place it on a white background to view the colour results.
• Check that the negative control tube has remained a pink/red colour. If the negative control has changed to a yellow or orange colour, then the results are compromised, possibly by cross-contamination. Either way, record the result on the results sheet. In the case of compromised results, the best action is to change gloves and thoroughly clean the area, the equipment and the outside of the sample tubes before repeating the entire set of controls and samples. If a second set of compromised results occurs, contact AgEtal for advice.
• Check that the positive control tube has changed to a yellow colour. If the positive control has not changed to yellow, then the results are also compromised, as this result suggests that the reagents have not worked. Either way, record the result on the results sheet.
• Then record the results for each sample. If the controls have worked correctly, then any sample that is orange should be treated as a possible weak positive result and can be sent to AgEtal for confirmation.