Hello,
I am doing recombinant cloning. for this purpose i have to cut plasmid and then gel purify the right size band for further procedure. but in my case, i am using TIANGEN gel extraction kit and after purification i m not getting sufficient amount of Plasmid. however i am using 2ug to 3ug concentration of plasmid for restriction digestion and band size are exactly right and i am cutting agarose neatly with less gel on it but still getting very low amount after purification. has anyone use TIANGEN kit before? and also is there any other way to purify desired band from gel?
Most of these kits bind denatured dna on a membrane and then elute in water or dilute tris buffer. You can usually improve the yield of purified dna by heating the elution buffer to 70c and leave it on the membrane for twice as long as the recommended time quoted in the kit and if necessary do another elution to make sure that all of the plasmid has been released from the membrane
Your are a little bit stingy with your DNA ! digest more DNA, load on several wells.... the yield of these kits also vary with the length of the fragment and salt concentration in the washing buffers.
Please digest more plasmid and load in a single lane. During elution use pre-heated water (65 ͦC) pour it at the center of the column and stay it for a few minutes.
( if it is an empty vector then you should get a single band)
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