Mini-Prep Plasmid Isolation and Identification

Page 3-53 in lab manual & handout

This is the second experiment (of a total of 3 experiments) for your molecular lab report.

This experiment has two components Mini-Prep isolate of plasmid DNA Identification of plasmid DNA by gel electrophoresis (next week)

Take cell and gently break them apart Precipitate cellular debris in pellet Save nucleic acids in supernatant Precipitate nucleic acids to a pellet Reconstitute pellet in either RNase or water* 37C 15-30 min. (longer the better) Gel Electrophoresis to confirm isolation *to be further discussed

Each group pick up the following tubes E. coli Cell Resuspension Buffer Lysis solution Potassium acetate Isopropanol Plasmid suspension1xTris Buffer RNAse or water ----------------------------------------- Loading dye

2. Isolation of Plasmid DNA: Add 350 ul of lysis solution to cells Gently mix, put on ice for 5 minutes

1. Centrifuge cells Discard supernatant Resuspend pellet in 200 ul resuspension buffer (tube-7) Incubate room temp. for 5 minutes

2. Isolation of Plasmid DNA Add 200 ul of potassium acetate to cell-lysis mixture Mix gently Place on ice 5 minutes Centrifuge (14,000) for 5 minutes

3. Isolation of Plasmid DNA Carefully remove 0.5 ml of supernatant, place in new tube. This contains your plasmid Add 1.0 ml ice cold isopropanol Place on ice for 10 minutes

4. Isolation of Plasmid DNA Centrifuge for 10 minutes You should now see a pellet of nucleic acids (plasmid DNA + RNA)! Odd number groups add 5 ul RNase and 5 ul Even number groups add distilled water Carefully remove & discard all supernatant Air Dry inverted for 10 minutes

5. Isolation of Plasmid DNA Add 60 ul of buffer to dissolve pellet Remove 40 ul of this dissolved solution and add 5 ul of loading dye To the remaining 20 ul of sample add 20 ul of Buffer and 5 ul of loading dye. Store in freezer for next week.

Next week Confirm your isolation with gel electrophoresis