15 Lab Protocol: DNA and PCR

Procedure 1: plasmid DNA isolation from bacteria

1. Work in a group of two.  Each group should obtain one Eppendorf tube containing a bacterial pellet (code name = A or B). Place in tube rack.

1. Add 200 µl of P1 Buffer to the tube and resuspend pellet completely by vortexing or pipeting.

1. Add 200 µl of P2 Buffer and mix by inverting the tube 2 – 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. Proceed to the next step within 1-2 minutes. Purpose of P2 buffer is to lyse the cells.

1. Add 400 µl of P3 Buffer and mix gently but thoroughly. Do not vortex. The sample will turn yellow when neutralization is complete. P3 buffer removes contaminating cellular debris as a pellet in step 6, and neutralizes the pH so plasmid DNA will attach to column in step 7.

1. Allow the lysate to incubate at room temperature for 1-2 minutes.

1. Centrifuge sample(s) for 2 minutes.

1. Place a Zymo-Spin™ IIN column in a Collection Tube and transfer the supernatant from Step 6 into the Zymo-Spin™ IIN column. When pipetting the supernatant be careful not to disturb the green pellet to avoid transferring any cellular debris to the column.

1. Centrifuge the Zymo-Spin™ IIN/Collection Tube assembly for 30 seconds.

1. Discard the flow-through in the Collection Tube, making sure the flow-through does not touch the bottom of the column. Return the Zymo-Spin™ IIN column to the Collection Tube.

1. Add 200 µl of Endo-Wash Buffer to the column and centrifuge for 30 seconds. Discard flowthrough.  The washes further purifies plasmid DNA

1. Add 400 µl of Plasmid Wash Buffer to the column. Centrifuge for 1 minute.

1. Transfer the column into a clean 1.5 ml microcentrifuge tube and then add 30 µl of DNA Elution Buffer to the column. Centrifuge for 30 seconds to release the plasmid DNA in the flowthrough.

Procedure 2: agarose gel electrophoresis to analyze plasmid DNA

(note: agarose gels contain the mutagen Ethidium bromide; therefore, gloves should be worn whenever handling the gel)

1. Transfer 5 mL DNA to a new Eppendorf tube containing 6 mL loading buffer/dye.

1. Load 10 mL of sample into well of 1% agarose gel (containing EtBr to allow visualization of DNA) submerged in 1X TBE running buffer.

1. Load control samples = pAMP and pKan

1. Once all wells have been loaded, connect electrodes (be sure positive electrode is on far side of gel from wells).

1. Turn on power and allow migration to occur for 30 minutes. Turn off gel.

1. Instructor (wearing gloves) will transfer gel to UV light box to visualize the results. If UV light box is not enclosed, students should wear UV protective glasses to view.

1. Determine which lanes of gel contain pAMP and which lanes contain pKan.

Procedure 3: Introduce plasmid DNA into E. coli cells – transformation

1. Scrape cells from petri dish using sterile pipet tip or toothpick.  Mix cells in 100 mL ice cold, 50 mM CaCl2 and place in ice bucket. Incubate for 10 minutes.

1. Add 2 mL plasmid DNA from procedure 1 and incubate tube 10 minutes in ice bucket.

1. Transfer tubes to 42o C water bath for 45 seconds (heat shock). Return tubes to ice bucket.

1. Add 300 mL of LB growth medium, mix gently, and incubate 30 minutes at 37o C to allow bacterial cells to recover and begin expressing the antibiotic resistance gene.

1. Spread 50 mL cells onto LB growth medium containing the antibiotic ampicillin or kanamycin.

1. Incubate plate overnight at 37o C to allow cells to divide and form a colony.

Note that bacteria with pAMP will grow (form visible colonies) on the LB + ampicillin plate.  pKAN contains a kanamycin resistance gene such that bacteria can grow on the LB + kanamycin plate.

Questions:

Were we successful in isolating plasmid DNA?  Explain.

Were we able to determine the identities of strains A and B by agarose gel electrophoresis? Explain

Predict what the LB + ampicillin and LB + kanamycin plates will look like after overnight incubation at 37o C if strain A contains pAMP and strain B contain pKAN. Use “+” to indicate growth and “-“ to indicate no growth on the plate diagrams below.

Materials required:

1. E. coli pellets in Eppendorf tubes (6 with strain A, 6 with strain B per section).

1. Zymogen Research plasmid miniprep kit (12 tubes per section)

1. Eppendorf tubes: 24 per section: 12 for elution of plasmid DNA and 12 for preparing electrophoresis samples

1. 1% agarose gels with EtBr (2 gels per section), electrophoresis chambers and UV visualization

1. LB agar plates with fresh E. coli colonies for preparation of competent cells (1 per section), Eppendorf tubes containing 100 mL 50 mM CaCl2 (12 per section), LB growth medium (5 mL per section), LB + Amp agar plates (3 per section), LB + Kan agar plates (3 per section).

1. P1000, P200, and P20 micropipettes and corresponding tips. (6 of each type of micropipette per section)

1. 500 mL beaker for waste tips, tubes and liquids.