IGEM:UC-Merced/2009/Notebook/E. hydro Express
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Beginning Tuesday November 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
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
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
7. JW1228-1/P1 culture was checked at 3 pm and there was a large amount of turbidity, which is opposite to what we should have seen. This means that potentially the P1 phage did not infect the JW1228-1 cells and instead, the JW1228-1 cells continued to grow through incubation. Due to this observation the JW1228-1/P1 culture was left to incubate until 4:30 pm on 11/15/12.
8. JW1228-1/P1 culture was removed from the incubator at 4:36 pm on 11/15/12. The JW1228-1/P1 culture appeared less turbid then when checked at 3 pm however, still more turbid then expected due to P1 phage activity.
9. Drops of chloroform were added to the JW1228-1/P1 culture and 3 ml of solution was centrifuged in each culture tube. Only 2 ml of supernatant was available from each 35 ml centrifuge tube. The supernatant was kept in 1 ml tubes and four tubes were collected.
10. The 1 ml tubes are labeled as CH AB 11/15 and kept in the 4°C refrigerator.