Hi all,
I need to screen a random library of genetic elements in mammalian cells after cloning, so I need to DNA purify >50 constructs.
I understand that the concentration of DNA from a miniprep is too low to use in transfection. I really don't want to spend the money to buy 50 midipreps and only use a fraction of the DNA for one transfection. It also doesnt seem very doable to concentrate a miniprep's worth of DNA via ethanol precipitation.
-Is there a way to purify small amounts of DNA cheaply and at high enough concentration to transfect with?
-Are there any other ways to concentrate miniprep DNA reproducibly?
-Anyone have any tricky cloning techniques that minimize mole bio work for library screening in mammalian cells that doesnt require sublconing the mammalian cells?
Actually you don't have to depend on kits, you can prepare your own plasmid extraction reagents following the Sambrook and Russell 2001 (Molecular Cloning: A laboratory manual). All you need are:
1) Alkaline lysis solution I
- 50mM glucose
- 25mM Tris-HCl pH 8.0
- 10mM EDTA pH 8.0
- autoclave and store at 4C
2) Alkaline lysis solution II
- 0.2N NAOH (freshly diluted from 10N stock)
- 1% SDS
- prepare fresh and use at room temp
3) Alkaline lysis solution III
- 5M Potassium acetate 60ml
- glacial acetic acid 11.5ml
- distilled water 28.5ml
- store at 4C
After get the culture pellet, resuspend in 100ul of Alkaline Solution 1 and vortex. Add 200ul Alkaline Solution II, invert mix, put on ice. Add 150ul cold Alkaline lysis solution III, invert mix and store on ice 3-5min (At this point, you can pool the tubes together to get higher yield). Centrifuge 5min 4C, max speed. After this you can purify using phenol chloroform method.
Actually you don't have to depend on kits, you can prepare your own plasmid extraction reagents following the Sambrook and Russell 2001 (Molecular Cloning: A laboratory manual). All you need are:
1) Alkaline lysis solution I
- 50mM glucose
- 25mM Tris-HCl pH 8.0
- 10mM EDTA pH 8.0
- autoclave and store at 4C
2) Alkaline lysis solution II
- 0.2N NAOH (freshly diluted from 10N stock)
- 1% SDS
- prepare fresh and use at room temp
3) Alkaline lysis solution III
- 5M Potassium acetate 60ml
- glacial acetic acid 11.5ml
- distilled water 28.5ml
- store at 4C
After get the culture pellet, resuspend in 100ul of Alkaline Solution 1 and vortex. Add 200ul Alkaline Solution II, invert mix, put on ice. Add 150ul cold Alkaline lysis solution III, invert mix and store on ice 3-5min (At this point, you can pool the tubes together to get higher yield). Centrifuge 5min 4C, max speed. After this you can purify using phenol chloroform method.
Hi,
I would still recommend Quiagen miniprep, but your choice. Go for Gibson cloning, its fast and efficient.
Start mini-prep with 10ml culture, with column., we get 8microgram DNA, sufficient to do all basic transfection, sequencing test.
Good luck.
Anand
i would recommend you to try The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs
Article The Miraprep: A Protocol that Uses a Miniprep Kit and Provid...
I would use a mini plasmid kit (We use Qiagen). They are efficient and consistent in quality. We routinely achieve >20 microg DNA from a 6 ml o/n culture at a concentration of ~500ng/uL (However amount is plasmid/insert/strain dependant). This is easily sufficient for sequencing/small-scale transfection. If you want more concentrated DNA then elute in smaller volumes.
I would like to suggest you that try to use DNA mini-prep Kit https://www.jenabioscience.com/molecular-biology/rna-dna-preparation-and-cleanup
I used it for long time and I think it can help you to prepare DNA sample at high enough concentration for downstream application.
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