I am cloning a vector from a 6 kb plasmid. I started with 10 ug plasmid. I did double digestion to remove 2kb fragment and then used phosphotase to treat the vector. After steps of digestion and purification, I wonder what the recovery rate should be? Considering the removal of 2kb, if 100% recovery, I should get 6.6 ug. I calculated I only have ~10% recovery. Is this normal?
Which purification did you do? For a 4 Kb plasmid, I'd guess that phenol/chloroform followed by ethanol should be around 90% (does not get rid of 2 kb fragment). Electroelution->precipitation is around 70%. Gel purification largely depends on the kit and the gel. 10% is a bit low. We only get that with >12kb fragments. Can you give more details of your protocol?
Thanks Anna for your answer. I used gel purification kit from Qiagen. Vector was purified through a spin column.
Hi, your recovery yield from the column is also dependent on the volume that you elute in. Try to elute in 20uL, and also do a 2nd separate elution in a new tube, and compare the yields.
Also make sure that you add the elution buffer directly on the filter of the column, leaving it at room temp for about 2 minutes before spinning down - it makes a huge difference.
Technically you should be able to retain about 80% of your starting concentration.
My answer is a bit latte but I can only tell you regarding this kit, that the addition of isopropanol (which iis optional in the kit) increase yield as well as prolonging the incubation step befooe elution. Prewarming of you elution buffer to 50°C is also advantageous
Regards
Sven
Thank you all for your help. I will take a note of that and consider these conditions in my next experiment. :)
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