FRAGMENT ISOLATION FROM LM AGAROSE |
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Last Update: December 2006 |
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| NOTES: | |
| 1. Most likely, this is your first step in the purification process right after a restriction enzyme digest. Use as much DNA as possible as a good portion of it (>50%) will be lost in the subsequent steps. >10 mg of DNA is my usual starting material. If other protocols are used to purify the DNA fragments (columns, ...) less DNA is necessary (each phenol extraction is only 50 to 70% efficient in recovering DNA) | |
| 2. If the digest will require an Alkalike Phosphatase (AP) treatment, it is good practice to treat with AP right before purification as well as right before phenol extraction to make sure all the fragment has been dephosphorylated. Therefore, if necessary, treat digest with Alkaline Phosphatase before starting this protocol | |
| PROCEDURE | |
| 1. Make 100 mL of 1.0% Agarose in 0.5-1% TBE and let it solidify in gel apparatus with an appropiate comb | |
| 2. Cut out portion of gel to be used for fragment isolation | |
| 3. Prepare ~50 mL of 1% Low Melting Agarose and load on cut out region of gel. Let it solidify at 4oC (alternatively, all the gel can be made of LM agarose) | |
4. Load samples and separate bands by running gel at 4oC (RT is also ok) |
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| 5. Cut bands out (visualization in UV box) and place them in a 1.5 mL eppendorf tube | |
| 6. Centrifuge samples for 10 secs and add 1X Vol (~200 mL) of TE buffer (can store agarose at -20oC if necessary) | |
| 7. Incubate at 65oC for 5 minutes, vortex into solution, incubate for 5 more minutes and vortex again (the idea of this step is to completely melt the LM agarose and leave as fluid a solution as possible (addition of extra TE buffer may aid in this process) | |
| 7.1 Alkalike Phosphatase treatment if necessary | |
| 8. Add ~300-400 mL of TE-saturated Phenol, incubate at 65oC (2-3 minutes) if phenol was cold, vortex and centrifuge for 3 minutes | |
| 9. Add ~300-400 mL of 24 Chloroform : 1 isoamyl alcohol, vortex until solution turns milky/clowdy (~30 secs) and centrifuge for 3 minutes | |
| 10. Precipitate DNA (upper phase) with 0.8X Vol of 2-propanol, 0.1X Vol of 3M NaOAc, pH 5.2, by mixing and centrifuging at 4oC for 30 minutes. Dry pellet in SpeedVac (5 miniutes) and resuspend in 20 mL of water | |