a. Lysozyme buffer once made should be used in 24 hrs. So once made, the solutio

Question

a. Lysozyme buffer once made should be used in 24 hrs. So once made, the solution should be kept on ______ or in the _________. b. Why not make a lysozyme stock of 5000 mg/ml (means 1000x) and freeze it so it lasts for months? ___________ c. What OD was used to monitor growth of bacterial cells? _________ Induction IPTG is abbrev for __________ You have been provided 1 M IPTG. You need final conc 1 mM IPTG to induce the bacterial cells. What should be your working solution of IPTG? __________ Make a 2 ml of working solution of IPTG: If you have a readymade IPTG solution of 200 mM, and you need to induce with 1mM IPTG, how much should you add to 50 ml cells? Show calculations.

Explanation / Answer

6. a) ice or -20C refrigerator.

b) it is difficult to solubilize 5000mg in 1ml. Secondly, lysozyme is a protein and upon continuous freezing and thawing will denature/degrade the protein during continuous usage.

c) optical density to monitor bacterial growth is 600nm. This is the wavelength of the light that is minimally absorbed by the bacterial cells.

7. Isopropyl -D-1-thiogalactopyranoside

1M IPTG = 1000mM IPTG. To make 1mM solution need to dilute 1000 times. For small culture volumes (2ml), such a working solution of 1000x would be creating errors to extract small volumes. Working solutions should always be calculated depending on the culture volume. For example, 1M IPTG should be fine for induction of 1 liter (1000ml) cells. However, for 2ml we need 0.001ml of IPTG which is error prone. For this make small working solutions as shown below:

2ml of working solution of IPTG: Initially, make 200mM from 1M IPTG (1000mM) as working solution: Since this is 5 times dilution (1000/200), add 0.4ml of 1M IPTG to 1.6ml of buffer (to make a final volume of 2ml).

8. Working stock - 200mM

In 50ml cells, the volume of IPTG should be 1mM which is 200 fold dilution (200/1). The required volume of IPTG is therefore: 50/200 = 0.25ml.

Take 0.25ml from 200mM working stock and add it to 50ml of cells (NOTE: Addition of 0.25ml to 50ml produces negligible change in volume)

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