Often this becomes a headache that we do not have enough yield of our #PCR or #RestrictionDigestion products to carry on with #cloning or other #downstream applications. Let me take you through a modified protocol in sync with the market leader kits #Qiagen, #MachereyNagel and #Sigma.
Protocol:
The very first thing is to run the gel smoothly at permissible voltage, DO NOT go over 120 volts please! This has been mentioned specifically in certain manuals.
While cutting the specific band, remember to give it as little UV exposure as possible. Use the 'Prep #UV' button on the omnipresent #BioRad gel documentation systems for cutting gel.
Weigh the excised gel portion and add 3 times the Gel melting buffer considering each mg of weight equivalent to a uL of the buffer.
After this there could be variations in the protocols of different kits with some suggesting addition of #Isopropanol. Nonetheless after passing the solution through spin column (capacity is generally <= 10ug), while adding the #WashBuffer or 70% #ethanol to the column, let it stand at room temperature for 2-5 mins after addition.
Thoroughly spin the column to pass any and all of the residual buffer and keep the column in contact to air for at least 1 min, so that any traces of ethanol can evaporate.
Add pre-warmed (~65C) nuclease free water or buffer of your choice to elute DNA.
Keep the column with #eluent at 65C dry bath for <= 5 mins and then spin at high speed to elute the DNA.
Hope this helps!
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