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

(Difference between revisions)
 Revision as of 20:44, 14 November 2012 (view source)← Previous diff Revision as of 14:29, 15 November 2012 (view source)Next diff → Line 27: Line 27: Beginning Tuesday November 13, 2012 Beginning Tuesday November 13, 2012 - 10/13/2012 + 11/13/2012 Generation P1 Phage Stock Generation P1 Phage Stock Line 41: Line 41: 25000 ug/ml (V1) = 250 ug 25000 ug/ml (V1) = 250 ug V1 = 0.01 ml = 10 uL  --> kanamycin to add to strain JW1228-1 V1 = 0.01 ml = 10 uL  --> kanamycin to add to strain JW1228-1 - Incubate kanomycin in LB broth until 11am 10/14/12 + Incubate kanomycin in LB broth until 11am 11/14/12 - 10/14/12 + 11/14/12 P1 Phage P1 Phage Line 59: Line 59: 25000 ug/ml (V1) = 250 ug 25000 ug/ml (V1) = 250 ug V1 = 0.01 ml = 10 uL  --> kanamycin to add to strain JW1228-1 V1 = 0.01 ml = 10 uL  --> kanamycin to add to strain JW1228-1 - Incubate kanomycin in LB broth until 10:30 am 10/15/12 + 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 10/15/12

## Revision as of 14:29, 15 November 2012

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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 10/15/12