Paul.xu at 16:29, 14 August 2018

2018-08-14T16:29:44Z

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== Ethanol Precipitation Protocol ==

1.  Run  small PCR DNA fragments obtained   on agarose gels.

2. Cut out DNA fragments and place in a sterile 1.5 mL microcentrifuge tube. 3.  Ground gel with  a metal rod and  then add  150 μL of TE buffer  4. Frozen  gel in tube using dry ice   5. Incubate  the tube at 72 °C for 3 min. 6. Immediately following incubation, spin the tubes  at 10,000 x g for 30 s at room temperature (RT). 7. Transfer the solubilized gel solution to a new sterile tube (1.5 mL) 8.  Add  1/10 vol of sodium acetate (3 M, pH 5.2) and 2.5 vol of cold ethanol (or isopropyl alcohol). 9. Incubate the tubes  overnight at −20 °C. Note: The incubation time for the precipitation depends of the initial concentration of DNA in the sample. 10. Spin at 16,000 x g for 20 min at RT 11.Wash with 600 μL of 70% ethanol (v/v)

12. Air dry and re-suspend by adding TE buffer or water to the desired volume. 13. Store  at −20 °C until further use.

== Compaction precipitation Protocol ==

Compaction precipitation also was compared with the well-developed commercial QIAamp Circulating Nucleic Acid Kit

The supernatant is recovered and compaction agent is added to a concentration of 2.5 mM. After 15 min incubation on an end-to-end rotator, the samples are again centrifuged at 16,000×g for 15 min. The supernatant is discarded and the DNA pellet is washed with 100 µl Wash Buffer (50% isopropanol, 300 mM NaCl, 10 mM MgCl2) and 100 µl 75% ethanol, with centrifugation at 16,000×g for 10 min after each wash. The pellet is then dried in a SpeedVac, before finally being dissolved in 20 µl 10 mM Tris-HCl, pH 7.4.

Purification of plasmid DNA using selective precipitation by compaction agents—A scaleable method for the liquid-phase separation of plasmid DNA from RNA. 

Spin down overnight  cultures  of  E.  coli (2  ml).

Add 0.25 ml P1

Add 0.25 ml P2

Add 0.5 ml N3

Spin.

Collect supernatant

Precipitate plasmid  by 0.7volume of isopropanol.

Resuspend  DNA  in 400 ul of 10 mM Tris HCl, pH8.0 

Add equal  volume  of 2.9 mM spermidine stock.

Vortex

Spin down pellet

Wash with 800 ul  50% isopropanol solution of 10 mM MgCl2, 300 mM NaCl, and 25 mM EDTA.  

[[Category:Protocols]]

Recover small DNA fragment from Gel - Revision history

Paul.xu at 16:29, 14 August 2018