Low DNA yield for Gecko V2 library prep?

I followed the protocol for Gecko library prep and achieved excellent transformation efficiency using Endura cells (lentiGuide-puro library A). But when I apportioned the bacterial pellet into 0.45 g aliquots as recommended by the protocol onto six maxiprep columns, the DNA yield was only 100-300 ug plasmid per each. I would expect about 1 mg of plasmid DNA per gram of bacteria, and I only achieved about a third of that (~0.33 mg plasmid / gram of wet bacterial pellet.)

Does anyone else have experience prepping this library?

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Navy Dhampirover 7 years ago

I received good yield for the A and B libraries, 1.23mg and 1.62mg. I used the NEB alpha electrocompetent cells with the biorad electropulsar and grew them on the very large bioassay plates, as the protocol says, I used the Qiagen Maxiprep kits

Red Pegasusover 7 years ago

How many prep columns did you use per plate?

Orange Titanabout 7 years ago

I am having similar problems with the amplification of GeCKO v2 library. Did you solve your problems already? And if so, could you please share your solutions.

Navy Manticoreabout 7 years ago

I got a pretty decent yield for the Mouse GeCKO v2 lib A.

I used the Endura cells for transformation and Cfu were really high. Then i snap freezer the pellet (1.45g wet pellet). In theory i was suppose to use around 3 maxi prep column , but i only had midi-prep kit ( Qiagen Midi plus). So i just used 5 midi columns and eluted the samples. Pooled the five preps and i got ~900ug/ul conc in a total vol of 1ml. I made virus and the titre was pretty good .

Hope this helps

Gold Impalmost 4 years ago

In this post you mentioned to use 3 maxi prep columns for 1.45 g wet pellet, and I think I got the same info on this forum from another post. However, based on qiagen protocol, a maxi prep is only good to handle 100 ml culture with high copy plasmid (GECKO V2 is high copy). Assuming 3g pellet / 1 liter culture, it would be 0.3 g pellet for 100 ml culture for one maxi prep (QIA TIP 500). Then, 1.45 g pellet would require 5 maxi prep columns. Am I right?

In the case of low-copy plasmid, however, it is 500 ml culture (1.5 g pellet) per maxi prep column.

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