The overall goal of this procedure is to visually demonstrate the steps for using the lookout mycoplasma PCR detection kit optimized to detect mycoplasma contamination. This is accomplished by first heating the samples to 95 degrees Celsius to inactivate any DNA molecules that could render the desired template unsuitable for PCR. Next, prepare the samples for PCR using the provided tubes that are pre-coded with nucleotides, primers, and internal control DNA.
The third step of the procedure is the temperature cycling under conditions carefully optimized to give the greatest sensitivity and eliminate non-specific amplification. Ultimately, results can be obtained to determine if the samples in question are contaminated with mycoplasma through evaluation by agro gel electrophoresis. The main advantage of this technique over existing methods is that the reagents are pre aquatic and high quality PCR tubes.
This makes it more convenient and efficient. Another advantage of this kit is that it requires less than two mycoplasma genomes per microliter of sample to detect contamination. When performing the procedure for mycoplasma detection, it is important to avoid any possible contamination where appropriate personal protective equipment to keep skin exposure to a minimum, ensure that all reagents used are free of contamination and maintain proper aseptic technique at all times.
The procedure can either be performed directly on cell culture supernatants or samples can be prepared for use at a later date. To prepare samples for later use place 100 microliters of each supernatant in a sterile amplification tube and incubate at 95 degrees Celsius for five minutes. After the incubation, the samples can be stored at two to eight degrees Celsius for up to one week.
Just prior to testing the samples, centrifuge briefly to pellet any cellular debris. To prepare samples for PCR first, determine the total volume of DNA polymerase rehydration buffer required for the reactions. One unit of DNA polymerase per reaction should be added to the appropriate volume of rehydration buffer, which will vary with the tac used in this demonstration jumpstart TAC D 93 0 7 will be used in five reactions, three samples plus one positive control and one negative control.
Place the calculated volume of DNA polymerase into a clean micro centrifuge tube and follow with the calculated volume of rehydration buffer. Mix the DNA polymerase rehydration buffer gently by flicking the tube. Do not vortex this mixture.
Use the transparent reaction tubes provided in the kit to prepare the negative control and samples. These reaction tubes already contain the nucleotides, primers and internal control DNA pipette 23 microliters of the previously prepared DNA polymerase rehydration buffer. Mix in each of the negative control and sample tubes.
Then add two microliters of DNA free water to the negative control and two microliters of each sample to the respective sample tubes. Mix the contents by flicking the tubes. Contents should not be vortex.
To prepare the positive control, use the pink reaction tubes provided in the kit. These reaction tubes already contain the nucleotides, primers and internal control DNA at 25 microliters of DNA polymerase rehydration buffer mix to the reaction tube. Mix the contents by flicking the tube contents should not be vortex.
Then incubate the positive control, negative control and sample tubes at room temperature for five minutes. After five minutes, transfer all the reaction tubes to a thermal cycler. For PCR, set the following conditions for 40 cycles.
94 degrees Celsius for 30 seconds. 55 degrees Celsius for 30 seconds and 72 degrees Celsius for 40 seconds. An activation step is not required when using Jumpstart tack.
Once the PCR cycles have completed, remove the samples from the thermal cycler and place them on ice to cool to four to eight degrees Celsius. The samples can then be loaded onto an agros gel for electrophoresis directly load eight microliters for each PCR reaction into a separate lane.Loading. Buffer and dye do not need to be added prior to loading samples because they're included in the reaction tubes.
Stop the electrophoresis after the tracking dye has migrated 2.5 to three centimeters. Representative results of detecting mycoplasma by PCR are shown in this image of an aros gel lane. Two is the negative control, which should show a band at approximately 481 base pairs.
The absence of the negative control band at 481 base pairs is a good indicator that the TAC used was not sensitive enough. Mycoplasma positive samples will show bands in the range of 270 plus or minus eight base pairs as shown in lanes three through six. The band will be heavy for highly contaminated samples and faint for lightly contaminated samples.
All samples contain an internal negative control to demonstrate that the PCR occurred as expected. It is normal to see the absence of the internal control on highly contaminated samples. An absence of a band on both the positive and negative range as shown in lane seven, is a good indicator that the samples are inhibited.
If the sample is inhibited. The PCR inhibitors can be removed by performing a DNA extraction. After watching this video, you should have a good understanding of how to perform all of the necessary steps outlined in the kit procedure, as well as interpret the results and troubleshoot when necessary.
