Difference between revisions of "Team:METU Turkey/Experiments"
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D)Precipitate, wash, and redissolve plasmid DNA | D)Precipitate, wash, and redissolve plasmid DNA | ||
10)Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the | 10)Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the | ||
− | eluted DNA and mix. Centrifuge at ≥ 15.000 xg for 30 min at +4℃. | + | eluted DNA and mix. Centrifuge at ≥ 15.000 xg for 30 min at +4℃. |
− | decant supernatant. | + | Carefully decant supernatant. |
− | 11) Wash DNA pelelt with 2ml room-temperature 70% ethanol and | + | 11) Wash DNA pelelt with 2ml room-temperature 70% ethanol and |
− | at ≥ 15.000 xg for 10 min. Carefully decant supernatant. | + | centrifuge at ≥ 15.000 xg for 10 min. Carefully decant supernatant. |
12) Air-dry pellet for 5-10 min and redissolve DNA in a suitable volume | 12) Air-dry pellet for 5-10 min and redissolve DNA in a suitable volume | ||
of buffer. | of buffer. | ||
Line 305: | Line 305: | ||
Plasmid Isolation Protocol: | Plasmid Isolation Protocol: | ||
Resuspend the pelleted cells in 250μL of the Resuspension Solution. | Resuspend the pelleted cells in 250μL of the Resuspension Solution. | ||
− | Transfer the cell suspension to a microcentrifuge tube. The bacteria | + | Transfer the cell suspension to a microcentrifuge tube. The bacteria |
− | be resuspended | + | should be resuspended |
completely by vortexing or pipetting up and down until no cell clumps | completely by vortexing or pipetting up and down until no cell clumps | ||
remain. | remain. | ||
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Centrifuge for 5 min to pellet cell debris and chromosomal DNA. | Centrifuge for 5 min to pellet cell debris and chromosomal DNA. | ||
Transfer the supernatant to the supplied GeneJET spin column by | Transfer the supernatant to the supplied GeneJET spin column by | ||
− | decanting or pipetting. Avoid disturbing or transferring the white precipitate. | + | decanting or pipetting. Avoid disturbing or transferring the white |
+ | precipitate. | ||
Centrifuge for 1 min. Discard the flow-through and place column | Centrifuge for 1 min. Discard the flow-through and place column | ||
back into the same collection tube. | back into the same collection tube. | ||
− | Add 500μL of the Wash Solution to the GeneJET spin column. | + | Add 500μL of the Wash Solution to the GeneJET spin column. |
− | for 30-60 seconds and discard the flow-through. Place the column back into | + | Centrifuge for 30-60 seconds and discard the flow-through. Place |
− | the same collection tube. | + | the column back into the same collection tube. |
Repeat the wash procedure using 500μL of the Wash Solution. | Repeat the wash procedure using 500μL of the Wash Solution. | ||
Discard the flow-through and centrifuge for an additional 1 min to | Discard the flow-through and centrifuge for an additional 1 min to |
Revision as of 15:15, 15 September 2015
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