Question

3.Walk me step by step how to pour 1% gels from scratch. 2 gels = 100mL

a.Make 1L of 1x TAE from a 50x stock

b.Weigh out the agarose for 2 gels

c.How much TAE to add?

d.Heat up obviously

e.Cool down

f.Add Ethidium Bromide, how much?

i.Also note a safety precaution

g.How much DNA do I load and how much dye to add?

h.What happens if there is no dye?

i.What voltage and time do you set?

4.Make a buffer from scratch

a.1L of 5M NaCl and 1L of 1M Tris (look up MW)

b.Once your stock solutions are made make 1L of 200mM NaCl and 20mM Tris (use water to fill to 1L)

Answer 1

3.

a. Mix 20 mL of 50X TAE stock to 980 mL of water.

b. Weigh 1 gram of agarose for 100 mL.

c. Add 100 mL 1X TAE to 1 gram of agarose.

f. Add 20 to 50 ?g of Ethidium bromide. [prepare ethidium bromide stock of 10 mg/mL in distilled water, and add 5 ?L from the stock to 100 mL agarose solution. If the stock concentration is less then add more volume to adjust the final concentration of ethidium bromide.]

g. For 5 ?L of DNA (total 20 ng or more), 1 ?L 6X gel loading dye. A total volume of 5-12 ?L mix can be added to each well.

h. DNA will not settle to the well. The progress of the cannot be visualised. Gel loading dye has glycerol which makes DNA sample denser to settle finely to the well.

i. 80-150 Volts, 1-1.5 hours.

4.

a. Dissolve 292 g of NaCl in distilled water and make up the final volume to 1 L to prepare 5M NaCl solution. Dissolve 121.14 g of Tris base in distilled water and make up the final volume to 1 L to prepare 1 M Tris.

b. Add 40 mL of 5M NaCl stock, 20 mL of 1M Tris, and make up the final volume to 1L using distilled water.