Preparation of Agarose Gel and Migration for PCR genotyping
1) Prepare migration buffer.
The migration buffers can vary and be TBE or TAE solutions.
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.
TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.
In molecular biology, TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation.
50ml to make the gel + 50ml of migration buffer => 100ml/gel.
2) Prepare the gel.
Prepare gel at 1.2% agarose (Agarose Electrophoresis Grade In vitrogen, Ref: 15510-027) in the chosen buffer => 0.6g/50ml/gel.
Dilute in a beaker, and microwave: 4x 30sec. Make sure it does not boil too much. Keep an eye on it, stop and shake it often.
When everything is well dissolved, put it under cold water until you can hold the beaker without burning yourself.
Add the safe DNA (Cambridge Biosciences GelRed 10,000X in water 0.5ml, Ref: IBRBT41003), d=1/10000.
Pour about 50ml in the plastic tank. Wait 10-15min for the gel to set. Remove the well-makers and add the migration buffer.
3) Load the gel.
5ul DNA Ladder
10ul PCR product
Run at 100V for 60min. Check black negative on top, and always check there are bubbles after plugging the cover.
Preparation of Agarose Gel and Migration for PCR genotyping —> PDF