• The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl.
•Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced.
• Technique: PCR
• The reaction tubes included with the kit are pre-coated with appropriate dNTPs, primers, and loading dye.
Upstream tips
• The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl.
•Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced.
Protocol tips
• Technique: PCR
• The reaction tubes included with the kit are pre-coated with appropriate dNTPs, primers, and loading dye.
Publication protocol
Cell culture supernatants can be tested directly or the sample can prepared for use at a later date. To prepare for later use place 100 μl of supernatant in a sterile amplification tube and incubate at 95° C for 5 minutes. Once this is complete the sample can be stored at 2-8° C for up to one week. Just prior to running the sample briefly centrifuge (5 seconds) to pellet any cellular debris. To prepare the samples for PCR, determine the total volume of Jumpstart Taq DNA polymerase/rehydration buffer required for the reactions. We will be preparing 5 total reactions. Five reactions will require 2.5μl of Taq and 114.5μl of rehydration buffer. This will contain a minimum of one unit of Taq per reaction and 22.5μl of rehydration buffer per sample reaction and negative control plus 24.5μl of rehydration buffer for the positive control. 1 unit of DNA polymerase per reaction should be added to the appropriate volume of rehydration buffer. This will vary with the Taq used. Place the calculated volume of Taq DNA polymerase into a clean microcentrifuge tube and follow with the calculated volume of rehydration buffer. The DNA polymerase/rehydration buffer should be mixed gently by flicking the tube. This mixture should not be vortexed. To prepare the negative control and samples use the transparent reaction tubes provided in the kit. The reaction tubes provided in the kit already contain the nuclueotides, primers and internal control DNA. 23 μl of Jumpstart Taq DNA Polymerase/Rehydration buffer mix, as prepared in the previous steps, should be placed in each of the negative control and sample tubes. Add 2 μl of DNA free water to the negative control and add 2 μl of the sample to each of the sample tubes and label. Mix the contents by flicking the tubes. Contents should not be vortexed. 5. To prepare the positive control use the pink reaction tubes provided in the kit. The reaction tubes provided in the kit already contain the nuclueotides, primers and internal control DNA. Add 25 μl of Jumpstart Taq DNA polymerase/Rehydration buffer mix, as prepared in the previous steps, to the reaction tubes and label. Mix the contents by flicking the tubes. Contents should not be vortexed. Incubate the negative control, positive control and sample tubes at room temperature for 5 minutes. 6. For the PCR to take place the samples need to be placed in a thermal cycler. When using JumpStart Taq an activation step is not required. Place the reaction tubes in the thermal cycler. The cycles should be set as follows: 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 40 seconds. These cycles will be run 40 times. 7. Once the PCR cycles have completed the samples should be cooled to 4-8°C by removing them from the thermal cycler and placing them in ice. When the samples have cooled they should be loaded onto the agarose gel for electrophoresis. Loading gel and dye are not necessary as they are already present in the reaction tubes. Directly load 8 μl for each PCR into a separate lane. Stop the electrophoresis after migration of 2.5 - 3.0 cm.
Check out relevant papers found by Labettor's AI that are relevant for performing Cell Culture Contamination Detection Kit Mycoplasma using LookOut® Mycoplasma PCR Detection Kit from Sigma-Aldrich.
Download the product protocol from Sigma-Aldrich for LookOut® Mycoplasma PCR Detection Kit below.
We haven't found the manufacturer protocol for this product yet.
Videos
Check out videos that might be relevant for performing Cell Culture Contamination Detection Kit Mycoplasma using LookOut® Mycoplasma PCR Detection Kit from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
Outsource your experiment
Fill out your contact details and receive price quotes in your Inbox