Applied Biosystems® real-time PCRシステムでマルチプレックス反応は可能ですか。また、何種類まで可能でしょうか。
Applied Biosystems® real-time PCRシステムではマルチプレックスアッセイに対応しています。現在弊社では通常1つのwellで2色までの蛍光色素の使用をサポートしております。
また、2色以上の蛍光色素を1つのwellで同時に検出することも可能です。ただ、そのためにはプライマー同士の競合反応を抑えるようにプライマー濃度の最適化をすることが必須です。
I am getting high background and smeared bands on my TBE-urea gels. What should I do?
Here are some suggestions for your experiments:
- The RNA samples may have been degraded by RNases. Use standard precautions to prevent contamination.
- Make sure samples are heated for 3 minutes at 70°C just prior to loading. If this is not done, the oligonucleotides will not be fully denatured, which may result in a smeared background.
- Be sure to vigorously flush urea out of sample wells just prior to loading the sample. Urea will continually seep from the gel into the well. Urea is very dense and will force the sample into a ball.
- A sample volume over 10 µL may result in smearing. This volume is less than may be used with agarose gels.
- If ultrapure water (18 milliohms) is not used, smeared bands and high background may result.
- Check to make sure the running and sample buffers have been prepared and diluted correctly.
- Make sure to run the bromophenol blue until it reaches the slot. Stopping the run short can result in less than optimal results.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
Can I use a sample buffer containing formamide with the Invitrogen TBE-urea system?
There are many sample buffer formulations used; however, we have found a distinct difference in the band appearance depending on the sample buffer composition. After evaluating urea, formamide, and various buffer systems, we found that the sharpest, flattest bands were obtained with a urea, Ficoll, and TBE buffer solution. Sample buffers made with formamide provided fuzzy, indistinct bands.
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
Can I stain my TBE gel or my TBE-urea gel? How?
Yes, for ethidium bromide staining, soak the gel in a 2 µg/mL solution of ethidium bromide in ultrapure water for 20 minutes. Destain by rinsing with three successive 10-minute rinses of ultrapure water. Visualize bands under UV light
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.
I am planning to use a TBE polyacrylamide gel for electrophoresis of my nucleic acid fragment. How do I know if the size of my fragment is suitable for a particular gel you offer?
To help you choose the TBE polyacrylamide gel that will provide optimal separation results for your nucleic acid fragment, we have designed gel migration charts for each gel type offered. Please check the TBE gel migration charts on page 74 of the Novex Pre-Cast Gel Electrophoresis Guide (https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf).
Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.