A. Isolation of Plasmids from Transformed Bacteria.

B. Restriction/Gel Analysis of Isolated Plasmids.

The goal of this experiment is to identify transformed bacterial colonies that contain the pCR-based plasmid constructs with your genes of interest. This will be done by growing bacteria from a single colony, isolating the plasmid DNA from these bacteria, and doing a restriction analysis to identify the plasmid that the bacteria contained.

Note: Alternatively, the alkaline lysis miniprep from week 1 can be used.

1. Solutions (will be prepared by TA’s):

All solutions for DNA work should be sterile to avoid DNase contamination.

2. (Will be done ahead of time by TA’s). Grow 0.5 mL cultures of E. coli overnight in 1.7 mL microfuge tubes placed in a flask for good aeration: 37°C/300 RPM.

3. Start a boiling water bath.

4. Harvest cells by spinning in microfuge (1.5 - 2 min./4°C). Discard spent medium and resuspend cells thoroughly in 0.2 mL STET by vortexing.

5. Add 20 µL 10 mg/ml lysozyme. RT/5 sec. 100°C (boiling water bath)/50-60 sec. Place on ice. Start one sample every 15 sec.

6. Spin in microfuge/4°C/10 min. Remove white pellet with broad end of sterile toothpick. Precipitate DNA by adding 0.2 mL isopropanol and mix by inverting 10X. -20° C/10 min. Spin for 3 min. Discard isopropanol. Rinse DNA pellet with 0.5 mL ice-cold 70% ethanol to remove isopropanol. Drain and dry pellet.

7. Resuspend DNA pellet in 0.1 mL TE. Spin for 2 min. and transfer the DNA solution to a fresh tube avoiding the small pellet. Add 6 µL 2.5 M NaCl. Add 0.2 mL ice-cold 100% ethanol. ^-80°C/5-10 min. Spin for 3 min. Discard ethanol. Rinse pellet with 0.5 mL 70% ethanol to remove NaCl. Drain and dry DNA pellet well.

8. Resuspend DNA pellet in 10 µL TE.

Restriction enzymes (HindIII) should be handled very gently. Any time the restriction enzyme stocks are out of the freezer (^-20° C), they should be kept on ice (4° C) since they slowly lose activity when they are warmer than ^-20° C. Also, never use the vortex to mix them; gently mix any restriction digestion(but not the enzyme stocks!) by gently tapping the tube with your finger or by drawing the solution slowly in and out of a pipet tip using a pipetman.

1. Cut 10 µL DNA with HindIII as described below:

Prelab Questions

1) An agarose gel is run to estimate the concentration of an isolated
plasmid. In one lane 6 ul of lambda Hind III was loaded at a
concentration of 33.3 ng/ul. In another lane a sample solution (1 ul
plasmid, 4 ul water, and 1 ul SAB dye) was loaded. After EtBr staining
a photograph is taken. The band intensity of the sample is most similar
to the intensity of the 6.6 kb fragment of the lambda Hind III. What is
the approximate concentration of the plasmid? [lambda Hind III gives
fragments of 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, 0.6, and 0.125 kb]

2) Another common method of checking the concentration of an isolated
plasmid is to the measure the Absorbance at 260 nm (lambda max for
DNA). If a 1:100 dilution of a plasmid sample yields an Absorbance of
0.2 at 260 nm, then what is the concentration of the sample? [Epsilon
260 for double stranded DNA is 50(ng/ul)-1 cm-1]

3) After calculating the concentration of a 2 kb plasmid to be 3.3 ug/ul
you realize that a procedure asks you to add 10 pmol. How much should
you add? [The MW of ds DNA = (660g)(length in bp)/mole]

Lab Report Questions

1. In lab, you isolated plasmids from two individual E. coli colonies and analyzed them using a restriction digest. Using the HindIII digest of bacteriophage l DNA that you ran as a marker on the gel, calculate the molecular weights of all of the products from your restriction digest. Remember that the relationship between gel mobility and molecular weight is roughly logarithmic. Make a plot.

2. Based on the products from your restriction digest, identify the plasmids that you isolated. Did you get fragments that you expected from the plasmids that you constructed? If not, what do you think happened?