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

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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
-
1. Overnight culture did not grow. Possible reasons are: (1) The JW1228-1 culture used was old and found in the 4°C refrigerator. It is possible that the culture was no longer living. To address this possibility we will redo the procedure using frozen stock.  
+
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 (procedure carried out as on 10/13/12)
+
2. Prepared new overnight culture with frozen JW1228-1 glycerol stock (following the procedure carried out on 10/13/12)
-
    a. LB
+
    a. LB
     b. Kanamycin stock 25mg/ml to add to strain JW1228-1
     b. Kanamycin stock 25mg/ml to add to strain JW1228-1
         C1V1=C2V2
         C1V1=C2V2
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 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 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.
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{{LnNotebookRecentChanges2}}
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-
|}
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 +
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.
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__NOEDITSECTION__
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10. The 1 ml tubes are labeled as CH AB 11/15 and kept in the 4°C refrigerator.
-
__NOTOC__
+

Revision as of 14:47, 16 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 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.

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