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

General Overview (some of these steps have been completed for you* 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 min. (longer the better) Gel Electrophoresis to confirm isolation *to be further discussed

Each group pick up the following tubes Tube 1 Tube 2 Tube 3 Tube 4A Tube 4B Tube 5 Tube 8 E. coli for DNA extraction Potassium acetate Isopropanol RNAse DI water Dilution buffer 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 Re-suspend pellet in 200 ul re-suspension buffer 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)! Carefully remove & discard all supernatant Air Dry inverted for 10 minutes Odd number groups now add 5 ul RNase and 5 ul Even number groups add distilled water

5. Dilution of Plasmid DNA Add 60 ul of buffer to previously dissolve pellet Remove 20 ul of this dissolved solution and add 5 ul of loading dye To the remaining 20 ul of sample +10 ul buffer and 10 ul of loading dye. Run gel!!

Next week Confirm your isolation with gel electrophoresis