Assignment: For each question, describe how you would make the solution using th

Question

Assignment: For each question, describe how you would make the solution using the stocks or reagents listed below.

On our lab prep shelves, we have the following solutions and powders:

Sodium citrate (FW = 43.7 g/mol)

SDS powder (FW = 288.38 g/mol)

0.5 M EDTA

IPTG powder (FW = 238.3 g/mol)

20 mg/mL X-gal solution

1000X ONPG

Premade LB

Premade LB agar

0.5 mM Colchicine

50X gel running buffer

Agarose powder

95% ethanol

100 mg/mL Ampicillin

10X BamHI restriction enzyme digestion buffer

milliQ water

dimethylformamide (DMF)

Example 1. (DNA isolation lab) How would you make 100mL of 0.15M Sodium Citrate, 5% SDS, 0.001M EDTA solution (one solution that contains all of these reagents)?

Answers

0.65 g Sodium citrate

5 g SDS

0.2 mL of 0.5 M EDTA

Add 75 ml of water and mix well. Then add water up to 100 ml to complete.

Example 2. (B-gal) How would you make a 10 fold dilution of 20 mg/ml X-gal? X-gal is not soluble or stable in water, but it is in dimethylformamide. What would the concentration of the diluted solution be?

Answer

Add 1 ml of the stock solution to 9 ml of DMF. 2 mg/ml

Problems for you:

1. (B-gal) How would you make 10 mL of a 0.1 M IPTG solution?

2. (hemoglobin) How would you make 250 mL 1X running buffer for your gel?

3. (B-gal) How would you make 48 ml of a 1X ONPG solution?

4. (B-gal) How wou50*ld you make 150 ml of a 100 mg/ml X-gal solution?

5. (restriction enzyme digestion). You are planning to do a 45 ml restriction digestion with BamHI. How much 10 X restriction enzyme buffer do you need to include?

6. (hemoglobin) How would you make 150 mL of 1.5% agarose (w/v) in 1X running buffer?

7. (hemoglobin) How would you make 7.5 L of 70% ethanol?

8. (transformation) How would you make 100mL LB agar + 100mg/mL Ampicillin?

9. (transformation) If we need 5.5 L of LB + 100 mg/mL Ampicillin for all three lab sections, how much total antibiotic do we need to order? Answer in mg.

10. (B-gal) How would you make a 4 fold dilution of X-gal? What would the concentration of the diluted solution be?

11. (DNA isolation) You need to read the absorbance of your DNA, but the DNA is too concentrated to get a good reading. You need to make a 10 fold dilution, but you do not want to dilute your whole sample. How would you make a 1 mL sample of your DNA to analyze?    

12. How would you make 2 ml of a 2 fold dilution of colchicine?   What would the concentration of the diluted solution be?

13. (B-gal) How would you make 500 mL LB + 40 mg/mL X-gal? (the LB is made, so you just need to figure out how much X-Gal to add)

14. (B-gal) How would you make 500 mL LB + 0.1 mM IPTG?

15. (B-gal) How would you make 500 mL LB + 40 mg/mL X-gal + 0.1 mM IPTG?

16. (B-gal) You need to make more of the 20 mg/ml X-gal stock, but somebody put an almost empty jar back on the shelf

Explanation / Answer

1) The molecular weight of isopropyl thiogalactoside (IPTG) is: 238.3. Thus, to make 0.1 M IPTG, we need to take 0.238 grams of IPTG in 10 ml of water.

2) To prepare 50 mL 1X running buffer for gel, we need to take

7.2 grams of glycine

1.51 grams of Tris base

0.5 grams of SDS

240 ml of water

Frist add 1.51 grams of Tris base and 7.5 grams of glycine together to 240 ml of water, then add 0.5 g of SDS. Finally, make up with 250 ml to get 250 ml of 1x running buffer for separation of hemoglobin.

3) To prepare 1x ONPG (13.27mM) solution we need to take 4mg/ml in 0.1 M NaPO4 pH 7.5.

4) To prepare 150 ml of a 100 mg/ml X-gal solution, we need to take 200 mg of X-Gal (MW = 408.63 g/mol) into 100 ml of polypropylene, and then add 10 ml of Dimethylformamide (DMF).

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