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.
[Parts Submission/Transformation] 1. Plated FMJ39 (5ul) with Streptomycin (20ul), for mini prep 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.
1. 0.5L of LB/CaCl2 was made.
A. 19.99g of LB agar was added to 500mL of water, to bring the total content of the Erlenmeyer flask up to approximately 500mL. B. 147g/mole CaCl2 * (0.0025 mole/ L) * 0.5L = 0.18375g CaCl2 C. 0.19g of CaCl2 was added to the 0.5 solution D. The 0.5L of LB/CaCl2 was separated into two Erlenmeyer flasks, each with about 0.25L in each.
2. 0.1L of Top agar LB/CaCl2 was made.
A. 2g of LB broth was added to 100mL of water, to bring the content of the Erlenmeyer flask up to approximately 100mL B.147g/mole CaCl2 * (0.0025 mole/ L) * 0.1L = 0.3675g CaCl2
3. Both were then autoclaved on the 15 minute cycle (setting 9).
A. Once out, the two 250mL flasks containing the LB/CaCl2 were put onto ice to cool. B. The top agar solution, once cooled, was labeled and left on the bench for future use. C. The solution is labeled as "CaCl2 Top agar 2/13/12"
4. The two 250mL flasks containing LB/Cacl2 were taken to a fume hood that was previously sterilized with 37% alcohol solution, for plates to be made.
A. The LB/CaCl2 solution was poured onto each plate and then the plates were swirled to allow for the solution to be evenly distributed over each plate. B. In total about 30 of these plates were left to sit with their tops off in the hood for 15-25 minutes to harden. C. Once the plates had hardened they were put back into the bag in which they came, labeled with a black and blue line, and then put into the walk in refrigerator. D. After about an hour of being left to harden, approximately 16 plates did not solidify and were then disposed of. This could be due to inadequate mixing of the original agar solution.
5. 100 mM Tris-Cl was prepared for a TM buffer.
A. 12.1g of Tris base was added to 100mL of water and stirred until the solution became clear. B. 5.3mL of HCl was added to the solution to bring the solution to a final pH of 7.38 C. The solution was labeled and left on the bench for future use.
1. Completion of 100mL of TM buffer.
A. 1.98g of MgCl2 was added to the 100mL of Tris-Cl that was prepared on 2/13/13 B. The concentrations of the Tris-Cl and the MgCl2 is 100mM each.
2/19/2013 (12:05 pm)
1. Bacteria for future use were cultured
A. 5mL of LB (prepared on 8/27 from lab bench) was placed into 14 ml round bottom tube. B. Stock JW-1228 was attempted to be retrieved from -80C freezer, but could not locate. C. Instead, JW-1228 + ku (Baij 11/14) from the 4C fridge was used D. JW-1228+ku was lightly vortexed because the cells had visibly aggregated to the bottom of the tube. E. 1uL of the JW-1228 was put into the 5mL of LB and then put into a 37C incubator overnight.
2. The TM buffer created on 2/15/2013 was autoclaved
1. Serial ten-fold dilutions were made with the phage stock and the TM buffer.
A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer. B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed. C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed. D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed. E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed. F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.
2. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.
A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer. B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates. C. The plates were then left to harden for about 45 minutes. D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates. E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.
3. The plates were left in the 37C incubator overnight.
1. The plates were retrieved from the incubator at approximately 1150 hours. There were no visible signs of phage plaques. We believe this was due to the fact that the top agar did not solidify as it should have. Solution: Prepare new top agar according to the composition listed in Current Protocols in Molecular Biology, "Top Agar" Unit 1.1
2. Preparation of the Top Agar
A. Directions listed for the to make 1 L of Top Agar from Current Protocols in Molecular Biology included: 10 g tryptone 5 g Yeast Extract 5 g NaCl 7 g Agar B. We prepared the Top Agar using the directions on the bottle because its composition was very similar to that listed in the protocol and we felt the difference was negligible. 3.7 g of Top Agar for 100 ul C. The flask used to mix the top agar was labeled "LB Top Agar 12/21/13" D. "LB Top Agar 12/21/13 was taken to autoclave Autoclave set to program 9 for liquid autoclaving of 15 minutes.