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miniprep and maxiprep - (Feb/19/2009 )

Is there any difference between miniprep and maxiprep except that you get more ug plasmid with maxiprep? Is there any difference in the procedure?

-minemin-

no there is no other difference.
miniprep is for few mL, you get around 10 g
maxiprep is for 100 mL , you get around 500 g

miniprep column are cheaper than maxiprep, so depending on what amount of plasmid you need, you will prepare mini, midi, maxiprep.

-little mouse-

minemin on Feb 19 2009, 02:55 AM said:

Is there any difference between miniprep and maxiprep except that you get more ug plasmid with maxiprep? Is there any difference in the procedure?


2ml verses 6L cell culture.

Firstly, oxygenation is more difficult, which if not compensated (using a bevelled flask or a flask 5x, ideally 10x volume of the culture) will result in E. coli going into mix acid fermentation earlier. This will result in changes in cellular biochemistry and overall lower cell density. From a plasmid point of view, you will get less plasmid than expected from simply scaling the volume. For growth of large BACs... >150kb, this will result in significant increase in BAC fragmentation..ie lower quality yields. From a protein extraction point of view... changes in cellular metabolism may in the worst case cause your overexpressed protein to "disappear", change from soluble to insoluble or expressed subtle changes in post expression modification.

From extraction point of view... it is harder to lyse the cell mass in a "gentle" manner. For maxipreps, I freeze thaw the cell mass, break it up on a vortexer and then use lysozyme to degrade the cell walls. Then I add alkaline lysis solution and give the lysis solution + cell mass a single gentle swirl and leave to stand for the cells to lyse.

This careful treatment more important when handling BAC (70kb), compared to small plasmids (10kb).

RNA - RNAse treatment is no longer cost effective on scales of maxiprep. LiCl percipitation is used to remove the RNA.

Resuspension of the DNA pellet is no longer so easy. I use a plastic dropper to resuspend my DNA gently. Over dry a maxiprep DNA pellet and you will never go home.

-perneseblue-

minemin on Feb 19 2009, 11:55 AM said:

Is there any difference between miniprep and maxiprep except that you get more ug plasmid with maxiprep? Is there any difference in the procedure?



DNA from midi, maxiprep kits are quite clean and of transfection quality compared to miniprep DNA.

-scolix-

perneseblue on Feb 19 2009, 03:28 AM said:

minemin on Feb 19 2009, 02:55 AM said:

Is there any difference between miniprep and maxiprep except that you get more ug plasmid with maxiprep? Is there any difference in the procedure?


2ml verses 6L cell culture.

Firstly, oxygenation is more difficult, which if not compensated (using a bevelled flask or a flask 5x, ideally 10x volume of the culture) will result in E. coli going into mix acid fermentation earlier. This will result in changes in cellular biochemistry and overall lower cell density. From a plasmid point of view, you will get less plasmid than expected from simply scaling the volume. For growth of large BACs... >150kb, this will result in significant increase in BAC fragmentation..ie lower quality yields. From a protein extraction point of view... changes in cellular metabolism may in the worst case cause your overexpressed protein to "disappear", change from soluble to insoluble or expressed subtle changes in post expression modification.

From extraction point of view... it is harder to lyse the cell mass in a "gentle" manner. For maxipreps, I freeze thaw the cell mass, break it up on a vortexer and then use lysozyme to degrade the cell walls. Then I add alkaline lysis solution and give the lysis solution + cell mass a single gentle swirl and leave to stand for the cells to lyse.

This careful treatment more important when handling BAC (70kb), compared to small plasmids (10kb).

RNA - RNAse treatment is no longer cost effective on scales of maxiprep. LiCl percipitation is used to remove the RNA.

Resuspension of the DNA pellet is no longer so easy. I use a plastic dropper to resuspend my DNA gently. Over dry a maxiprep DNA pellet and you will never go home.


By the way, what is the aim of Midi- and Maxiprep ? is it for protein extraction or plasmid extraction ? if it is for plasmid extraction, for which application would one need such a quantity of plasmid ?

-Biog-

Biog on Feb 21 2009, 03:07 PM said:

By the way, what is the aim of Midi- and Maxiprep ? is it for protein extraction or plasmid extraction ? if it is for plasmid extraction, for which application would one need such a quantity of plasmid ?


well for my lab... it is primarily for plasmid extraction.

Midipreps are used to obtain what is effectively a permanent supply of a certain plasmid, either for transformation, or for latter modification. My lab also keeps its plasmids in DNA form rather than in cells.

Maxipreps are used to grow low copy plasmids and BACs. My lab makes BACs to create artificial centromeres for study. We follow the ground up approach using large BACs to carry our artificial centromeric sequence. The process requires a fair bit of BAC DNA as it is fairly inefficient.

-perneseblue-

You can only isolate DNA with these methods, for protein purification ypu may need HPLC or column cromatopgraphy.

And the method you need to use, depend how you will work with the DNA, or how many samples you have, if you are testing a recent clonning expreriment, you have many colonies to try, so you need to use a miniprep, if you need clean DNA for clonning or a blorring assay, the option are maxiprep, you have more DNA to work and it could be washed with EtBr and phenol for purification, and this method could be useful for enzime restriction assay.

-Dj Vu-

i think the method you need to use, depend how you will work with the DNA, or how many samples you have, if you are testing a recent clonning expreriment, you have many colonies to try, so you need to use a miniprep, if you need clean DNA for clonning or sequencing, then you need to do a maxiprep, becouse you have more DNA to work and it could be washed with EtBr and phenol for purification, and this method could be useful for enzime restriction assay.

-Dj Vu-

I think maxiprep is usually to extract transfection grade plasmid (to transfect into animal cells or ?plant cells?...not sure about the latter) because those maxi prep kits (like from Qiagen) would remove endotoxins probably due to their column and washing solutions, so the plasmid would have low endotoxin levels. Endotoxins are toxins found in the bacteria and may lead to cell cytotoxicity during transfection.

Miniprep usually doesn't remove any endotoxin and the plasmids extracted are usually used for analysis or for transformation.

-lab_member-

Mini or Maxi prep amount of DNA is depend upon the silicon packed in the column and if you endotoxin removal solution then it will be more quality than normal preparation.

-bochum-