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

(Difference between revisions)
 Revision as of 14:29, 15 November 2012 (view source)← Previous diff Revision as of 23:07, 29 November 2012 (view source) (→Notes)Next diff → (7 intermediate revisions not shown.) Line 64: Line 64: 1. JW1228-1 overnight culture grew. The next step in the procedure was carried out according to Yale University. 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 2. Preparation of 10ml containing 20% Glucose solution a. Calculations a. Calculations 20g/100 ml = x/10ml 20g/100 ml = x/10ml x=2 g of glucose 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. 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 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 a. Calculation to make a 1:100 dilution of JW1228-1 overnight culture Line 76: Line 79: b. Two culture tubes were created for P1 phage infection 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 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 + + 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. + + + 11/28/12 + + [Parts Submission/Transformation] + 1. Plated FMJ39 (5ul) with Streptomycin (20ul), for mini prep 11/29/12 + + 11/29/12 + + 1. FMJ39 plate showed transformation through appearance of red spotting along edge of one quadrant of plate. + 2. Mini prep performed on DNA. Tube labeled "FMJ DNA" placed in -20 degrees C freezer. + 3. Insufficient TAE buffer to run gel - will reattempt tomorrow 11/30/12.

## Revision as of 23:07, 29 November 2012

Search this project

## Description/Abstract

• Place short description of project or notes regarding this project
• Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Donec porta commodo tellus. Nam a est eget libero mollis tincidunt. Aliquam purus. Quisque nulla ligula, facilisis in, pulvinar sed, molestie a, quam. Vestibulum at pede. In in sem eget odio eleifend placerat. Phasellus ultricies felis quis sapien. Etiam molestie volutpat quam. Praesent pulvinar scelerisque mi. Nam mi urna, fringilla eu, mattis sed, venenatis id, nunc. Maecenas velit eros, congue ut, placerat in, ornare vel, sem. Aenean porta enim sit amet felis gravida posuere. Phasellus faucibus nibh et orci.

## Notes

• Place some notes here for visitors
• Example: Next team meeting on Tuesday, June 18th! Be there!

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

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.

11/28/12

[Parts Submission/Transformation] 1. Plated FMJ39 (5ul) with Streptomycin (20ul), for mini prep 11/29/12

11/29/12

1. FMJ39 plate showed transformation through appearance of red spotting along edge of one quadrant of plate. 2. Mini prep performed on DNA. Tube labeled "FMJ DNA" placed in -20 degrees C freezer. 3. Insufficient TAE buffer to run gel - will reattempt tomorrow 11/30/12.