DNA cleaning during cloning

Reagents

During molecular cloning, you may need to clean the DNA, remove salt and enzyme from previous step or change the buffer system for enzymatic digestion, etc. Using the column provided in DNA extraction kit, DNA can be efficiently cleaned and retrieved to TE buffer in 5 min.

1. Based on the volume of your DNA reaction, add 5X (v/v) PB buffer to the DNA and mix by pipetting. PB buffer is provided in QiaPrep Mini plasmid kit as a washing reagent.

2. Load the DNA to a column from DNA extraction kit.

3. Spin down at 14K RPM for 1 min at RT.

4. Add 700 ul buffer PE from DNA extraction kit to the column.

5. Spin down at 14K RPM for 1 min at RT.

6. Discard the flowthrough and re-spin down at 14K RPM for 1 min at RT.

7. Transfer the column to a clean eppendorf tube and add 30 ul buffer TE to the center of the column. Leave on bench for 1 min.

8. Spin down at 14K RPM for 1min at RT.

9. Collect the DNA in a new eppendorf tube.


Comments

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Posted by neverthink (nevernetbug) Tue, 23 Dec 2008 15:42:33
Subject: Another simple way to clean your DNA
For cloning, DNA purification with Any column is too much as far as I am thinking. Simply buy fishtank fiber(also called glasswools) and make an easy filter yourself, takes 2-5 mins and you done with all handling and can go on with you PCR, sequencing, ligation or whatever.

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