A. Ligation of DNA from Restriction Digests.

B. Gel Analysis of Restriction Digests.

C. Transformation of Bacteria with DNA from Ligations.

The goal of this experiment is to create pCR-based plasmids containing the inserts that you cut from the pU29-based plasmids and to transform E. coli with these newly contructed pCR plamids.

1. T4 DNA ligase should be treated gently as in the case of the restriction enzymes: keep the ligase stock on ice (when it’s not in the freezer) and mix the ligation reaction by gently tapping the tube with your finger. Do not vortex.

Mix the following in a microfuge tube and incubate at room temp for 1 - 2 hrs:

8 µL sterile ddH₂O

2 µL 10X Ligase Buffer

9 µL DNA from your restriction digests (H350 ng pCR or pCR-a^-)

1 µL 3 U/µL T4 DNA Ligase

20 µL

10X Ligase Buffer:

500 mM	Tris-HCl, pH 7.5
70 mM	MgCl2
10 mM	Dithiothreitol (reductant)
10 mM	ATP

2. Add 80 µL TE. Add 6 µL 2.5 M NaCl. Add 0.2 ml ice-cold 100% ethanol. Mix by inversion. ^-80 °C/5-10 min. Spin 10 min. at 4°C to recover DNA pellet. Rinse drained DNA pellet with 0.5 ml 70% ethanol to remove any NaCl still left. Drain and dry pellet thoroughly. Resuspend DNA thoroughly in 10 µL TE.

1. TA’s will demonstrate how to prepare an agarose gel. A 0.7% agarose gel will be prepared and used by the class.

2. Load 6 µL 33.3 ng/µL l HindIII digest as a MW marker.

3. Add 6X SAB to 1X (2 µL of 6X SAB) to a 10 µL aliquot of your sample before running the gel.

4. Run 0.7% agarose gel in 1X TBE at H35 V/5 min. and then, at H80 V/1 hr.

5. Stain with 5 µg/ml ethidium bromide in water for 15 min. with gentle (50 RPM) shaking. Destain for 5 min. with gently shaking.

6. Photograph at f4.5/2 sec. l HindIII digest will give bands at 23.1, 9.4, 6.6, 4.4, 2.3, 2.0., and 0.56 kb.

6X SAB: Sample Application Buffer (for agarose gels)

6X	TBE
30% (v/v)	Glycerol
0.25%	Bromophenol Blue
0.25%	Xylene Cyanole FF

10X TBE:

108 g	Trizma Base
55 g	Boric Acid
40 ml	0.5 M Ethylenediamine Tetraacetic Acid, pH 8.0 (chelating agent)
to 1 L.	dd H2O

This protocol is designed to be used with either fresh or frozen-competant cells made by the rubidium choride method, though works well with standard CaCl competant cells as well (with reduced efficiencies).

1. For each plasmid that you are using to transform bacteria, 200 µl of competant cells are added to a 5-mL Falcon polypropylene tube (on ice). You will need 3 tubes. One for your experimental plasmid and two for a pUC19 (Amp^r) and a pCR control.

2. Add 2 µL of your isolated plasmid (H 40 - 80 ng) to the cells and gently swirl. Consult your TA for the appropriate volume of pUC19 to add.

Note: It is important to keep the amount of DNA below 50 ng, or the efficiency of transformation will actually decrease. For transforming supercoiled plasmid, 1 ng is plenty.

3. Incubate the DNA with the cells on ice for 30 min.

4. Heat shock the cells by transfering the tubes to a 42 ÚC waterbath for exactly 2 minutes.

5. After the 2 minute incubation, transfer the tubes to a 37Ú C waterbath and add 2 ml of 2 x YT medium to each tube.

6. Incubate for 1 hour at 37ÚC.

7. During this incubation, remove the plates from the coldbox, and allow to warm up to room temp.

8. Plate out each transformation twice. Once without concentration (step 9) and once with concentration (step 10).

9. After the 1 hour incubation, spread 100-200 µl of cells onto one plate with a flamed spreader. Generally this will yield sufficient colonies. However, if this is a blunt-end ligation, or particularly large inserts are being employed conentrating the suspension may be desired (see step 9 below).

10. To concentrate the suspension, put 1000 µL of suspended cells in a 1.5 mL microfuge tube and spin at 2900rpm/4min (600 – 800 g). Dump off all but ~100-200 µl of the supernatant, and resuspend the pellet with a pipetman. Plate this concentrated suspension as above.

11. After the plates are dry, incubate them inverted O/N at 37ÚC for 12-16 hours. (Longer times will result in satelite colonies).

1. Why do you have to add Sample Application Buffer (6X SAB) to your restriction digest before loading it on the agarose gel?

2. What reagent will you use to visualize your DNA bands on the gel? Are there any safety procedures that you need to follow when using this reagent?

3. Which antibiotic should you use in your plates to select for bacteria containing your pCR-based constructs? Which antibiotic should you use in your plates to select for bacteria containing the pUC plasmid control?

1. Draw a map of the products that you expect for each of your ligation reactions.

2. In the transformations that you performed, which plasmids would you expect to confer ampicillin resistance to E. coli ? Which would you expect to confer tetracycline resistance to E. coli ? What would grow on plates with no antibiotics?

3. Analyze the bands on the gel analysis of your restriction digest from week 1. Did you get the products that you expected? What products did you expect? Explain your reasoning. If you did not get the products you expected, what products do you think you got? Explain your reasoning.