Plasmid purification for Gibson Assembly - (Aug/09/2016 )
I am Yu, working in biology and biotechnology.
Now I have already obtained plasmid with an amount of 4 ug. What I gonna do is to double digest this 26 kb plasmid with XbaI and BamHI. Finally I will get two fragments, one 22 kb that I will take for my Gibson Assembly vector, and trash the other 4 kb. But I am facing with a problem. I have no idea about plasmid purification, that is to say I do not know how to separate enough amount of purified 22 kb vector. If run on agarose gel, I am not sure the High Pure PCR Purification Kit (Roche) will work well on this frament with so big size. Does anyone have good and detailed protocol on big plasmid purification? Can share your instruction will be so helpful.
Thanks in advance!
PH:IAC purification the digested fragments and then continue your Gibson assembly. The digested fragments could not combine again and only overlap with your objective fragments during Gibson assembly process...
good luck .
I agree with Hongying 100%, but if you have a real need to purify a large fragment in the future Qiagen and other companies make kits for this purpose.
Here is Roche's protocol for purifying large fragments (>30 kb) using their High Pure kit: https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_3_05_p9-11.pdf