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Protocol

Double Digestion |Gel Extraction|Miniprep | Chemical Inducible Expression of GFP |DNA Purification |Ligation of Insert DNA | PCR |Preparation of Competent Cells| Site-directed Mutagenesis| Transformation|

Protocol for DNA Purification from Reaction Mixture

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Here is a suggested protocol; this protocol can be used to purify a wide range of DNA fragments with recoveries of >80%. The bolded should be noticed for a nice DNA extraction.

1. Put EB (elution buffer) or ddwater at 65 degree water bathing.

2. Add a 3:1 volume of Binding Buffer to the reaction mixture (e.g., for every 100 ul of reaction mixture, add 300 ul of Binding Buffer). Mix thoroughly.

Check the color of the solution. A yellow color indica tes an optima l pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

3. Pour the solution to a fresh adsorption column. Centrifuge at 13000rpm for 1 min.

Pour off the liq uid in the collection tube. For critical samples, repeat the operation above.

4. Add 600 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min. Pour off the liq uid into beaker.

5. Centrifuge at 13000rpm for 10 min to spin the ethanol down.

6. Put the column into a fresh EP tube. If necessary air-dry the pellet for 10-15 min to avoid the presence resid ual ethanol in the purified DNA solution. Resid ua l of ethanol in the DNA sample ma y inhibit downstream enzymatic reactions.

7. Add 30-50 ul elution buffer (EB) to elute the DNA.

8. Get 5 ul of the eluted sample to identify with electrophoresis.

Note:

1. If a large amount of DNA is purified or if the volume of the binding reaction is greater tha n 1.5 ml increase the incubation time of the binding step to 15 min.

References:

• Current protocols in molecular biology