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Got problem with maxiprep - (Dec/01/2008 )

Hi guys

I am new here today and I already have some questions

I made a clone with pcDNA3-FLAG plasmid and it went well in miniprep but in maxi i only got very low DNA yield

It might be the gene I cloned that is toxic to E coli but empty plasmid only with FLAG sequence also results same

I use nucleobond maxi kit but it does not seem something is wrong with the kit because 1ml miniprep from overnight maxi culture gives low DNA yield

It must be something during the culture but i have no idea

I made LB freshly everytime, I also changed ampicillin, and even i did chloramphenicol method but nothing works

This drives me crazy somebody help me!!


One more thing, I found that in pcDNA3.1 origin of replication was changed to pUC

It was ColE1 in pcDNA3

Do you think there was something wrong so Invitrogen changed ori?

-MetallicAngel-

What volume of cells are you using, and how long are you growing your cells for? What does the culture look like before prepping?

-Ginger Spice-

QUOTE (Ginger Spice @ Dec 1 2008, 11:41 PM)
What volume of cells are you using, and how long are you growing your cells for? What does the culture look like before prepping?

500ml of LB culture. The cell is DH5a FYI. I usually grow cells to 4ml culture from single colony overnight and inoculate 1ml into 500ml culture then culture o/n again. I'm not so sure how it is supposed to look like. I did not measure OD but centrifuged weight of cell pellet was nearly 3g from 500ml culture.

-MetallicAngel-

Is overnight 12 hours or 16 hours or 18 hours? This makes a big difference. If you grow your cells too much they will die on you, so I normally do a 12 hour incubation, and dilute the starter culture 1:1000 (i.e. 500 ┬Ál in 500 ml). This gives me good yields with high purity.

Another big difference between doing a mini and a maxi is that you are using very big purification columns. It is important to ensure that all your buffers are correct (for example, some wash buffers require that you add ethanol to them) the columns are well equilibrated before you bind DNA, and that all traces of ethanol are removed from the column before elution.

-Ginger Spice-