Standard practice for plasmid minipreps is to grow ~5 mL ecoli cultures overnight (~16 hrs). This works well and I always get good plasmids.
However, a few times I wanted just *a little bit* of plasmid. So I only incubated for a few hours:
2 mL cultures for ~7-8 hrs at 37C.
The culture is a bit cloudy by this point and gives an okay yield of plasmid DNA.
However, this plasmid DNA is always seems very poor quality.
If I submit it for sanger sequencing, the results are a little ugly. As if I have a couple SNPs. A tiny bit of contamination. The plasmid is there, but it is dirty.
If I try to electroporate it into bacteria, it always "arcs." Like there is very high salt contamination.
I must re-transform it into ecoli and do a proper, 16 hour overnight culture to regain good plasmid. Then the sequencing is clean and perfect, and it electroporates with no arcing.
Why is this? Why do dilute, low-volume cultures always give such bad plasmid DNA? I would have thought "young" culture would be cleaner.
It is very frustrating because short miniprep cultures seemed so nice and convenient. But the prep is so dirty I think I am wasting my time.
Are you using a column prep kit for this or just the base solutions?
I agree with your logic but, as with many things, logic can change based on understanding. When I read your statement, it made me think more deeply about the process of plasmid purification.
When you purify the plasmid from a young culture, the amount of DNA is limited in the sample (remember that the kits are designed for a certain number of cells to be processed). This means that the column in this case has a binding capacity that is nowhere near saturation, potentially allowing other salts to bind (think about why DNA binds them - the charge is critical) and be "purified" with your sample. This would explain the arcing that you see when you try and transform the plasmid.
I am not an expert on sequencing but have to believe that the salt contamination would also mess with the analysis.
My suggestion, if you want to experiment, would be to grow a larger volume for the shorter time (maybe several tubes or flasks) and test the preparation from one versus a pooled sample. If the pooled sample has less trouble electroporating, my suggestions are probably correct and you can send it out for sequencing as well.
Are you using a column prep kit for this or just the base solutions?
I agree with your logic but, as with many things, logic can change based on understanding. When I read your statement, it made me think more deeply about the process of plasmid purification.
When you purify the plasmid from a young culture, the amount of DNA is limited in the sample (remember that the kits are designed for a certain number of cells to be processed). This means that the column in this case has a binding capacity that is nowhere near saturation, potentially allowing other salts to bind (think about why DNA binds them - the charge is critical) and be "purified" with your sample. This would explain the arcing that you see when you try and transform the plasmid.
I am not an expert on sequencing but have to believe that the salt contamination would also mess with the analysis.
My suggestion, if you want to experiment, would be to grow a larger volume for the shorter time (maybe several tubes or flasks) and test the preparation from one versus a pooled sample. If the pooled sample has less trouble electroporating, my suggestions are probably correct and you can send it out for sequencing as well.
Aiguo Xu
260/280 and 260/230 ratios are ~2.0.
I determined plasmid quality by:
1. Quality of sanger sequencing of plasmid
2. Ability to electroporate plasmid into bacteria
Sequencing:
The plasmids do sequence *okay,* it's just not very clean.
Good plasmid sequencing result: ~1000-1200 bp read. No uncertain nucleotides.
Bad plasmid sequencing result: 600-800 bp read. ~1% of nucleotides are wrong call.
I want to do 7-8 hr miniprep for cloning, but this low quality sequencing is not sufficient for cloning.
When I re-transform the plasmid and do 16 hr miniprep, sequencing is perfect.
Electroporation:
16 hr miniprep: Elute into 50 uL. Perfect electroporation with 5 uL
7-8 hr miniprep: Elute into 50 uL. Always arc with 5 uL. Like high salt contamination.
to electroporate "dirty" DNA we used to dialyse the miniprep using small nitrocellulose filters (0,025um VSWP01300 from millipore) by floating the filter on milliQ water in a petri dish; put a drop of your DNA for 30min (the drop will be bigger) and then electroporate. Do it in a quite corner of the lab... do not move the petri dish !!!
OD260 on minipreps ("by hand" ie without purification on a column) is useless there is so much contaminating RNA and protein ...
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