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Choose Right Agarose Gel Concentration to Efficiently Separate PCR DNA Fragments

How to Choose Right Agarose Gel Concentration to Efficiently Separate PCR DNA Fragments

Did you experience that it is difficult to separate two similar sized DNA fragments on agarose gel? Some researchers found that it is not so easy to judge whether they get two similar PCR bands or not, especially for researchers who are doing mouse genotyping. The easiest way is to re-design PCR primers to amplify much different sized PCR DNA lengths. But sometimes it is not an optional for detecting small deletions in a given genomic DNA fragment. At this time you may adjust the agarose gel concentration to separate DNA fragments.

Which buffer should I use, TAE or TBE buffer? First, agarose gel running buffer can affect DNA fragment separation. The commonly used running buffers are Tris-Acetate-EDTA (TAE) and  Tris-Borate-EDTA (TBE). TBE is a better conductive buffer than TAE for longer running, which can avoid overheating during electrophoresis. TAE buffer gives improved separation of large DNA fragments. On the other hand, TBE buffer is better to separate <2kb fragments. If your down application is to do enzymatic related applications, such as, enzyme digestion, gene cloning, etc please don’t use TBE since borate in TBE buffer inhibits many enzymes.

What agarose gel should I use? High concentration or low concentration? Please refer the following table to see the best DNA fragment separate range.

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Recommended Agarose concentrations for optimal resolution separation.

GEL (%) OPTIMAL SEPARATION RANGE (bp) RECOMMENDED BUFFER
0.75 20,000 – 500 bp 1X TAE
1 16,000 – 300 bp 1X TAE
1.25 10,000 – 250 bp 1X TAE
1.5 5000 – 200 bp 1X TAE
1.75 2500 – 100 bp 1X TAE
2 1500 – 50 bp 1X TAE
GEL (%) OPTIMAL SEPARATION RANGE (bp) RECOMMENDED BUFFER
0.75 12,000 – 500 bp 1X TBE
1 8000 – 300 bp 1X TBE
1.25 4000 – 200 bp 1X TBE
1.5 3000 – 150 bp 1X TBE
1.75 2000 – 100 bp 1X TBE
2 1000 – 50 bp 1X TBE

 

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