IGEM:UC-Merced/2009/Notebook/E. hydro Express

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Lab Notebook

Beginning Tuesday November 13, 2012

11/13/2012

 Generation P1 Phage Stock

  1. Prepared P1 Stock from Yale University
   a. LB
   b. Kanamycin stock 25mg/ml to add to strain JW1228-1
       C1V1=C2V2
      25000 ug/ml (V1) = 50ug/ml (5ml)
      C1 = 25000 ug/ml  --> kanamycin concentration
      C2 = 50 ug/ml   
      V2 = 5 ml broth
      25000 ug/ml (V1) = 250 ug
      V1 = 0.01 ml = 10 uL   --> kanamycin to add to strain JW1228-1 
     Incubate kanomycin in LB broth until 11am 11/14/12

11/14/12

P1 Phage

1. Overnight culture did not grow. Possible reason is: The JW1228-1 culture taken from the 4°C refrigerator was old and was probably no longer living. To address this possibility we will redo the procedure using frozen JW1228-1 stock. 2. Prepared new overnight culture with frozen JW1228-1 glycerol stock (following the procedure carried out on 10/13/12)

   a. LB
   b. Kanamycin stock 25mg/ml to add to strain JW1228-1
       C1V1=C2V2
      25000 ug/ml (V1) = 50ug/ml (5ml)
      C1 = 25000 ug/ml  --> kanamycin concentration
      C2 = 50 ug/ml   
      V2 = 5 ml broth
      25000 ug/ml (V1) = 250 ug
      V1 = 0.01 ml = 10 uL   --> kanamycin to add to strain JW1228-1 
     Incubate kanomycin in LB broth until 10:30 am 11/15/12

11/15/12

1. JW1228-1 overnight culture grew. The next step in the procedure was carried out according to Yale University.

2. Preparation of 10ml containing 20% Glucose solution

    a. Calculations
        20g/100 ml = x/10ml
        x=2 g of glucose

3. The 20% glucose solution was made using filtered water and 2 g of glucose. The resulting 10 ml of glucose solution was filtered.

4. Preparation of Overnight JW1228-1 culture to be used in P1 phage infection

    a. Calculation to make a 1:100 dilution of JW1228-1 overnight culture
         1/100=x/5ml
         100x=5ml
         x=0.05 ml or 50 ul
    b. Two culture tubes were created for P1 phage infection
              i. Each tube include: 50 ul of overnight culture, 50 ul of 20% glucose, and 25 ml of 1M CaCl2

5. JW1228-1 overnight culture prepared for P1 phage solution to be incubated until 11:55 on 11/15/12

6. 20ul of P1 phage was added to JW1228-1/P1 solution after incubation. It will be incubated until 11/15/12 at 3 pm