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Minnesota/14 July 2008
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|NOTE: Used Buffers to purify or washout any cell debris so only has DNA. Used spin column b/c the column binds to DNA (has DNA affinity) and all other solution will drain through. | |NOTE: Used Buffers to purify or washout any cell debris so only has DNA. Used spin column b/c the column binds to DNA (has DNA affinity) and all other solution will drain through. | ||
|- | |- | ||
| - | |2. '''Spectrophotometry:''' | + | |2. '''Spectrophotometry:''' 'Spec' purified preps to check concentration of DNA in the ligated products. |
| + | |- | ||
| + | |3. '''Double Digest:''' | ||
Revision as of 16:27, 14 July 2008
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| 1. Plasmid DNA Purification Using the QIAprep: Use 2mL LB cultures of ligation products from 07-11-2008. Procedure: |
| a. Resuspend bacterial cells in 250uL Buffer P1 and transfer to microcentrifuge tube |
| b. Add 250 uL Buffer P2 and invert 4-6 times |
| c. Add 350uL Buffer N3 and mix immediately by inverting 4-6 times |
| d. Centrifuge for 10 minutes @ 13,000 rpm in a table-top centrifuge. White pellet will form (made up of cell debris). |
| e. Apply supernatants from step D to the QIA prep spin columns. Centrifuge for 30-60 seconds --> discard the flow through. |
| f. Wash the QIA prep spin column by adding 0.5mL (500uL) Buffer PB. Centrifuge for 30-60 seconds. Discard the flow through. |
| g. Wash QIA prep spin column by adding 0.75mL (750uL) Buffer PE. Centrifuge for 30-60 seconds. Discard the flow through. |
| h. Centrifuge for additional 1 minute to remove residual buffer. |
| i. Place the QIA prep spin column in a clean 1.5mL centrifuge tube. To elute DNA, add 50uL of Buffer EB (10mM Tris-Cl, pH 8.5) to center of spin column and centrifuge. |
| NOTE: Used Buffers to purify or washout any cell debris so only has DNA. Used spin column b/c the column binds to DNA (has DNA affinity) and all other solution will drain through. |
| 2. Spectrophotometry: 'Spec' purified preps to check concentration of DNA in the ligated products. |
| 3. Double Digest: |
