Question

OMPLETE & REMOVE THIS PAGE. HAND IN WHEN YOU ENTER THE LAB. FOR CALCULATION PROBLEMS, PUT A BOX AROUND YOUR ANSWER. TA Signature (at beginning of class) Name: WORKSHEET FOR DAY 5 (DUE AT BEGINNING OF LAB) 1. (a) What is the purpose of taking the A600 of all 20 of your transformed cultures in section N? (b) Why do you need to take the A600 of all 20 of your cultures before doing the p-gal assays? 2. What is the purpose of the SDS and chloroform in the p-gal assays? 3. Why do you need to add the ONPG at 15-20 second intervals when doing the B-gal assays? Below is a duplicate of the table provided in Part N. Complete the table so that you can prepare your master mix. Copy these numbers into part N since you will have to turn in this sheet in the beginning of lab. X5 reactions (master mix) Per reaction Colony DNA water 10x buffer 22yL 1.25mM dNTP mix 1OUL 4 M primer #1 4uM primer #4 6.25UL 6.25UL polymerase0.5UL total SOJL 0.5uL

Answer 1

1.A. A 600 is the absorbance required to have a countable colonies around 10*6 cfu/ml

1.B. it is necessary to get countable colonies . Too less or too high population will not give an the true number of transformants

2. They are used for cell permiation

3.the presence of the B galactosidase enzyme will give a yellow colour on acting on The substrate

4.DNA colony

Water 110microL

10x buffer 25 microL

DNTP mix 50ML

Primer 1 31.25 ML

Primer 2 31.25ML

Pol 2.5ML

Total 250ML