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Flow Chart Unit 1 - Plasmid Purification: 150 ul cells in lysis buffer on ice

You must have a rubber pipette bulb, a lab coat, and safety glasses. Lab coats and protective eye wear are REQUIRED for the experiments that use phenol. Please do not wear shorts or sandals because phenol causes severe chemical burns when it contacts skin; wash with water to remove phenol.

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Flow Chart Unit 1 - Plasmid Purification: 150 ul cells in lysis buffer on ice

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  1. You must have a rubber pipette bulb, a lab coat, and safety glasses. Lab coats and protective eye wear are REQUIRED for the experiments that use phenol. Please do not wear shorts or sandals because phenol causes severe chemical burns when it contacts skin; wash with water to remove phenol.

  2. Flow Chart Unit 1 - Plasmid Purification: 150 ul cells in lysis buffer on ice + 60 ul lysozyme; mix 20oC,10 min + 200 ul SDS/NaOH; invert gently 0oC, 5 min + 150 ul potassium acetate; invert gently 0oC, 10 min label tube with seat number centrifuge 10 min @ 14,000 rpm transfer supernatant solution to clean tube & discard pellet; label tube with seat number

  3. Flow Chart Unit 1 – page 2: supernatant solution in clean tube, labeled + 10 ul RNase A; mix 20oC, 10 min + 500 ul phenol/CHCl3/isoamyl alcohol in hood vortex centrifuge 3 min @ 14,000 rpm transfer top phase to clean labeled tube in hood & discard tubes + bottom phase in organic waste + 10 ul 3.5 M Na acetate; mix + 500 ul ethanol; mix 0oC, 10 min centrifuge 10 min @ 14,000 prm

  4. Flow Chart Unit 1 – page 3: discard ethanol & retain pellet (DNA) centrifuge 10 sec @ 14,000 rpm remove residual ethanol with yellow tip dry pellet dissolve pellet in 50 ul TE label 2 tubes: seat # & “Pst” or “uncut” + 3 ul of 10x reaction buffer to each tube + 10 ul water to “Pst” & 12 ul water to “uncut” + 15 ul DNA to each tube + 2 ul PstI enzyme to “Pst” tube 37oC, 60 min store remaining plasmid DNA in freezer box; TAs will freeze digested DNAs

  5. Flow Chart Unit 1 - Agarose Gel Electrophoresis: retrievePstI-cut and uncut plasmid DNAs from -20oC centrifuge 15 sec label two new tubes “Pst” and “uncut” add 15 ul of DNA to each; (store rest on ice for transformation) + 2 ul loading solution to each tube; mix load into wells of gel (assigned by TA) indicate contents of wells on loading sheet TAs will load size standards in specified lanes connect leads (black @ top; red @ bottom) apply 80-100 volts until dye nears bottom I will photograph gels under UV light & post photos

  6. Flow Chart Unit 1 - Transformation: label 3 sterile tubes “cut,” “uncut” and “no DNA” add 50 ul competent cells to each; on ice + 5 ul of PstI-cut or uncut DNA hold on ice 40 min incubate @ 37oC, 20 sec hold on ice 2 min + 0.95 ml broth to each incubate shaking @ 37oC, 60 min prepare dilutions (see p. 52) spread on L+Amp agar incubate 30oC overnight check plates the following afternoon

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