purification of digested plasmids - (Jan/15/2006 )
Hi.
I just wonder if there is any method to purify restriction digested plasmids other than running digests on the low melting point agarose gel→cut out the target band→purify the plasmids with kits (e.g. Promega DNA purification system)?
1. Remove gel slice contain DNA fragment and place in 10 volumes of:
300 mM NaOAc, pH 7.0........300 ml 1 M NaOAC, pH 7.0
1 mM EDTA..........................2 ml 500 mM EDTA, pH 8.0
...........................................698 ml ddH20
2. Incubate at 22 C for 30 min. Transfer gel slice to a fresh tube.
3. Place tube in a Dry Ice/Ethanol bath for 5 min.
4. Puncture the bottom of a 0.5 mL microcentrifuge tube with a needle. Place the gel slice into this tube. Place this tube inside a 1.5 mL microcentrifuge tube.
5. Centrifuge for 15 min.
6. Collect the Eluent from the 1.5 mL eppendorf tube. Extract and precipitate the DNA.
OR :
DNA Fragment Purification from Agarose or Acrylamide
For fragments from 200 bp to 10 kb the agarose purification is ideal. For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred.
Solutions
Crush and Soak Solution
500 mM NH4OAc 3.3 g NH4OAc
0.1% SDS 0.1 g SDS
0.1 mM EDTA 20 ml 500 mM EDTA
up to 100 ml with Q
store at room temperature
3 M NaOAc pH 5.2
24.6 g anhydrous sodium acetate
pH to 5.2 with acetic acid and bring up to 100 ml with Q
store at room temperature
Other Reagents
DMCS treated glass wool (Alltech Assoc. Inc. #4037, 50 g)
0.22 mm disposable micro tip filters (syringe type)
blue tips with melted tips to serve as pestle for crushing acrylamide
Procedure
agarose gels
• Prepare spin columns by cutting off the cap of a 0.5 ml eppendorf tube and forming a hole in the bottom with a hot 18 ga needle. Fill this "mini-column" with a small ball of DMCS treated glass wool and pack down with a pipet tip.
• Cut out the desired band from an agarose gel and place in a spin column inside a 1.5 ml eppendorf tube with the top cut off.
• Spin at 6,000 rpm in a microfuge for 10 minutes.
• Phenol/chloroform extract the flow through and EtOH precipitate with glycogen or tRNA and 10% v/v of 3M NaOAc pH 5.2.
• Wash and dry, resuspend in 20 microliters TE, run 10 microliters on a gel and use 1-2 microliters for a ligation.
acrylamide
• Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band.
• Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar."
• Add 1 ml crush and soak solution and incubate overnight at 37° C.
• Spin in the microfuge for 10 minutes at 14,000 rpm. Remove as much liquid as possible and add another 500 microliters of crush and soak solution.
• Repeat the spin and pool the recovered supernatant.
• Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above).
• Spin as usual, wash and dry. Resuspend in 20 microliters TE.
I just wonder if there is any method to purify restriction digested plasmids other than running digests on the low melting point agarose gel→cut out the target band→purify the plasmids with kits (e.g. Promega DNA purification system)?
This is the protocol I employ:
The digestion mixture (50 ul) is carried to 200 ul and an equal volume of phenol:chloroform 1:1 is added. This mixture is centrifuged 13000 rpm/5 minutes. To 175 ul of the acuose phase 400 ul of cold ethanol 95% and 20 ul of sodium acetate 3M pH 5.2 are added. 13000 rpm/20 minutes. To the pellet add 800 ul of cold ethanol 70%. 13000 rpm/10 minutes. Conserve the pellet and let it dry; after that, disolve the DNA in 20 ul of water. Run 5 ul in an agarose gel to stimate concentration.
I have good results, except when the sodium acetate solution has been taken from the freezer, so keep it at root temperature.
Hope this can be useful.
this thread might be useful as well:
Check this!
Thank you all. These are very helpful!