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Revision as of 22:21, 17 October 2014
Preparing/Autoclaving A media - 6/10/2014
A media:
Made 750 mL of A media
Weighed:
9g Pancreatic Digest, 9 g Difco Yeast Extract, 3 g Dextrose, 3 g KH2PO4 monobasic, 0.75 g MgSO4 * H2O, 750 mL distilled water
Poured into a plastic beaker and stirred for 40 mins.
Then placed in refrigerator at 4°C for 40 mins.
Poured 75 mL aliquots of media into 200 mL bottles.
For 200 mL storage bottle (Before autoclave):
- Put water in the container that will hold all the aliquoted bottles
- Put foil on lid of aliquoted bottles
- Open the lids of the bottles slightly but not completely
- Put autoclave tape on the foil
To autoclave:
- Use heat-resistant gloves to place container that contains bottles in autoclave
- Close autoclave with foot pedal
- Set up a username and password
- Choose liq60 = 1hr. and 32 mins.
- Wait for loud sound which means that the autoclave is compressed
- Take back cart and write down what time you started autoclave
After autoclave:
- Put on normal and heat resistant gloves
- Slide the container with bottles onto cart
- With heat gloves on, close the lid of each bottle and place outside container
- Get rid of water in container
- Place bottles back in container
- Wait for bottles to reach room temperature
- PLACE LABEL ON EACH BOTTLE WITH: NAME OF MEDIA, “AUTOCLAVED”, DATE, and NAME or INITIALS.
Culturing P. Acnes – Day 1 – 6/17/2013
For cultures at 24 °C:
Started with 15 mL of P. Acnes in enriched clostridial broth.
Pipetted eight 1mL aliquots of the liquid culture into eight 125 mL glass bottles near a flame.
Tightly closed lids of bottles.
Then individually opened autoclaved A media and aliquoted glass bottles of P. Acnes liquid culture near flame.
Poured approximately 30 mL of A media into each glass bottle.
Tightly closed bottle.
Put in autoclaved stirbar wrapped in aluminium foil into bottle without touching the stirbar.
Then placed bottle in 1.3 L LockandLock container.
Placed anaerobic detector in chamber.
Loosened the lid of glass bottle, placed anaerobic Gaspak in chamber and quickly closed lid.
Placed bottle on Corning stirplate at value of 4.
This was repeated with 3 other glass bottles.
For cultures at 37 °C:
Started with 15 mL of P. Acnes in enriched clostridial broth.
Pipetted eight 1mL aliquots of the liquid culture into eight 125 mL glass bottles near a flame.
Tightly closed lids of bottles.
Then individually opened autoclaved A media and aliquoted glass bottles of P. Acnes liquid culture near flame.
Poured approximately 30 mL of A media into each glass bottle.
Tightly closed bottle.
Placed two bottles in each 1.3 L LockandLock container.
Placed anaerobic detector in chamber.
Loosened the lid of glass bottle, placed anaerobic Gaspak in chamber and quickly closed lid.
Then placed container on a shaker in a 37 °C room.
There were two 125 mL bottles in each chamber.
Stored excess liquid culture in a centrifuge tube at room temp.
Created solution of ½ mL of P. Acnes liquid culture and ½ mL glycerol in microcentrifuge tube and placed in -80 °C freezer.
Possible errors: We did not use pipette tips that had been autoclaved L for transferring 1 mL of P. Acnes liquid culture into glass bottles.
6/18/14 – Plates for P. Acnes ATCC_11827 culturing
7 out of the 8 cultures are still pretty clear. All the anaerobic indicators in the chamber were still pink which means that the chambers are still anaerobic and the LockandLock is working well.
Possible source of error: One of the glass bottles in the 37°C rooms anaerobic chambers is much cloudier than the others. Carlos said this is fine and is most likely the result of us getting a clump of bacteria when we added it to the media.
We made 100 mL of A media for plates with this adjusted recipe:
| Component | 100 ml |
| Pancreatic Digest of Casein A | 1.2 |
| Difco Yeast Extract | 1.2 |
| D+ Glucose (Dextrose) | 0.4 |
| KH2PO4 monobasic | 0.4 |
| MgSO4*7H2O | 0.1 |
| Difco Agar | 1.5 |
dH2O 100 mL
Firstly, we mixed everything except the agar in an Erlenmeyer flask covered with aluminium foil. This was stirred on a stirplate for 40 minutes. We then placed it in a -4°C refrigerator for 32 minutes. After taking it out of the fridge, we put in our agar. The flask was then placed in a bin with water and autoclaved for 1hr. and 2 minutes.
Using water bath:
- Press set and wait
- Then while holding down set, increase temperature
After the A media came out of the autoclave, we tried to place it in the water bath at 55°C for ½ hour, however the flask floated in the water bath so we let it cool at room temperature. We let it cool for too long so it was no longer completely homogenous. We put it in the microwave for a period of 10 seconds four times.
Procedure for creating plates:
- Put on gloves
- Wipe down space with ethanol
- Turn on fisher burner
- Place plates around the burner
- Spray some ethanol onto a tissue and only handle autoclaved flask with this tissue
- Take foil off autoclaved flask with media which should be at 55°C or cool enough to hold without burning your handv
- Flame top of the flask for a couple of seconds
- Open lid of petri dish individually and pour on media until petri dish is ½ to 2/3 full
- Then close lid most of the way but leave a little bit open
- Repeat this for the other three petri dishes
- Let the petri dish cool for at least half an hour or until the condensation on the lid has completely disappeared.
- ENSURE THAT THE MEDIA IS SOLID
- Then close lid and seal the petri dish with a strip of Parafilm.
- Label the plate with date it was created, the media used, and name.
Possible source of error: We had a stir bar in the flask which we autoclaved. This fell into one of the petri dishes. We used two scoopulas, which were flamed with ethanol, to take the stirbar out of the petri dish. We also then flamed the media in the petri dish using the fisher burner. This petri dish was marked with an X.
6/19/2014 – ATCC_11827 strain cultured
We put in 1 mL of reinforced clostridial media into the ampule of P. Acnes, which we received from ATCC, using a syringe. Then, 700 µL of the resuspended P. Acnes was syringed into a microcentrifuge tube. The tube was opened next to the fire and 200 µL of the bacteria was pipetted into a 250 mL glass bottle. We also poured approximately 20 mL of A media into the glass bottle. This bottle was placed in a LockandLock with a Gaspak.
Liquid Culture
We put in 1 mL of reinforced clostridial media into the ampule of P. Acnes, which we received from ATCC, using a syringe. Then, 700 µL of the resuspended P. Acnes was syringed into a microcentrifuge tube. The tube was opened next to the fire and 200 µL of the bacteria was pipetted into a 250 mL glass bottle. We also poured approximately 20 mL of A media into the glass bottle. This bottle was placed in a LockandLock with a Gaspak.
Plates
- Put on gloves, wipe table with ethanol, turn on flame.
- If using plate spreader, dip in 90% ethanol and flame. If using loop, heat platinum loop until red hot and let cool for 30 secs – 1 min.
- If using platinum loop, after cooling touch onto gel and make sure it is cool and does not melt plate.
- If using plate spreader, open plate next to flame and pipette 50 µL into the center of the plate. Then spread around the plate using a mini rotating bench.
- If using platinum loop, then dip loop in suspended bacteria and streak on plate.
- Close petri dish when not streaking.
We made four plates.
The first plate was done with a plate spreader using 50 µL of bacteria. This was placed in a LockandLock with a Gaspak in the 37°C room on the shaker.
The other three plates were streaked. Two of these plates were stored in a LockandLock under anaerobic conditions in the 37°C room on the shaker.
The last plate was stored under aerobic conditions in the 37°C room on the shaker as a control.
Leftover ATCC_11827
We still had 450 µL of ATCC_11827 left in the microcentrifuge tube. This was mixed with 450 µL of 50% glycerol and stored in a -80°C freezer.
6/20/2014 – Got dam- E. Coli strain
We got the dam- E. Coli strain and placed in the -80°C freezer
Two of the glass bottles in the 37°C room were very cloudy and the rest were still somewhat cleara
In the 24°C room (lab), the cultures definitely have growth but not as cloudy as in the 37°C room.
6/23/2014 – Got dam- E. Coli strain
Plates with Erythromycin:
| Component | 200 ml |
| Pancreatic Digest of Casein A | 2.4 |
| 2.4 | |
| D+ Glucose (Dextrose) | 0.8 |
| KH2PO4 monobasic | 0.8 |
| MgSO4*7H2O | 0.2 |
| Difco Agar | 3.0 |
| dH2O | 200 mL |
Made a 50mg/ml stock solution of erythromycin in ethanol
After autoclaving the plate media put in 200 microlitres for every 1ml
So if making 200 ml then 40 microlitres of erythromycin
Username: grab
Pwd: 6428
Phenol: Chloroform Extraction
We used the protocol Carlos gave us.
O/D for most turbid strains
Most turbid strain had an O/D of 1.6. We decided that this would be used for further P. acnes strain 6919 cultures because it was too saturated.
The O/D of the second most turbid strain was 0.556. This was a good O/D for adding glycine. We added 0.75 mL of 20% glycine into our 30 mL culture as a cell wall weakening agent.
We also now have a stock solution of E. Coli transformed with the ampicillin resistance. In addition, we have a glycerol stock of the transformed DNA.
6/24/2014
Took the O/D of two of the cultures in the 37°C room. One was 0.354, the other was 0.202. Not ready for glycine yet. The cultures in the lab are not ready for glycine yet. We also got lysozyme and figured out how to get the glycerol.
6/25/2014
Took the O/D of two of the cultures in the 37°C room. One was 0.707, the other was 0.262. We added 0.50% glycine (0.75mL). The other culture was not ready for glycine. We also checked the O/D of the culture in the 37°C room which was previously at 0.556. It had actually decreased to 0.515.
We got the DNA back from the Phenol:Chloroform Extraction. We have to run it on a gel to determine if there is a lot of RNA present.
Made the Cheong buffer but did not autoclave. The recipe was for 272 mM sucrose + 7 mM sodium phosphate + 1 mM MgCl2. We decided to make 500 mL of the Cheong Buffer. This meant we added 46.552 g sucrose, 0.49686 g sodium phosphate, and 0.0476 g MgCl2.
6/26/2014
We made a 5 mL liquid culture of transformed E. Coli. We added 5 microliters of ampicillin to the liquid culture. This was left in a refrigerator for 14 hours overnight.
We added 20 microliters of TE buffer to the plasmid that Carlos gave us from the P/E extraction because there was too little of it present.
We nanodropped this sample and got a concentration of 0.2946 ng/microliter.
Making the gel
Need 25mL of media for the gel.
Should be 1X TE buffer
Concentration of agarose is 0.8 g/100 mL
Pour all of it into the flask and put towel stopper on flask. Microwave for around 1 minute in order to melt all the agarose.
Add ½ microliter of ethidium bormide (in red microcentrifuge tube)
Make sure comb is in. Pour into well.
Running the gel
Once the gel had set we added around 250 mL of TX buffer. The wells are connected to the black cord (negative). We added 1 microliter of the ladder in the first well, 5 microliters of pNIT linear plasmid + 1.25 microliters of loading dye in the second well, and 5 microliters of pBres36a plasmid + 1.25 microliters of loading dye in the third well. This was run at 110 kV for 1.5 hours.
Result of gel
When we checked the results of the gel, we were able to determine that the size of the pBres36a plasmid was 8 kb and that there was no RNA present as a result of phenol:chloroform extraction. However, the pNIT linear plasmid was not visible on the gel.
Took the O/D of the culture in the 37°C room which was 0.707. It was now 1.065, which means that it is ready for electroporation. We did an electroporation protocol (std 6/run13) for this culture. However, we did not add plasmid yet because we did not have enough. The cells were stored in the -80 room.
6/27/2014
We made TGE buffer for electroporation.
6/30/2014
We made to more cultures of ATCC_6919. We added 1 mL of the overgrown solution to 35 mL of A media. The O/D of the stock culture was 1.686. This was placed in the 37°C room
7/1/2014
We did a two phenol:chloform extractions yesterday. One was with E. Coli grown in A media. The other was with E. Coli grown in LB media. We thought we didn’t have success with the extraction with the E. Coli grown in A media. We did get DNA in the extraction, with the E. Coli grown in A media.
7/2/2014
We did three phenol:chloroform extractions with E. Coli grown in A media.
We also checked the O/D of two cultures of ATCC_6919. One was at 0.737 the other was at an O/D of 0.765. We ran 14/9 (std/run) and 10/2 on the two respectively. This meant we added 1.5 mL of glycine to both.
7/3/2014
Diluted 30 µL of DNA with 20 µL of TE 1X buffer.
DNA Sample #1 – 2 µg/µl
DNA Sample #2 – 3.2 µg/µl
DNA Sample #3 – 4.5 µg/µl
O/D’s of the two cultures today - 0.997, 0.971
DNA Sample #3 – 3.0 µg/µl – 80 µL total
DNA Sample #1 – 0.76 µg/µl – 160 µl total
We conducted two electroporations today. Std/run 10/2 and Std/run 14/9. We used DNA sample 1. Both arced. We suspect it might be because of the Cheong buffer, which contains magnesium chloride (a salt).
7/7/2014
We conducted four electroporations today. Std/run 10/2, Std/run 14/9 (twice), and Std/run 2/1.We used DNA sample 1. Std/run 10/2 and Std/run 14/9 (twice) both arced but Std/run 2/1 did not. So we could conclude that the Cheong buffer was the reason for the arcing.
We also made 6 A media plates without erythromycin.
7/8/2014
We made A media
Did electroporation on 1/8
Started new liquid cultures:
2 11827 upstairs
1 6919 upstairs
3 11827 downstairs
1 6919
7/10/2014
Plated 1/8.
7/11/2014
Checked the OD of all of the liquid cultures in 37 C. 3 of them had a high enough density to add glycine. They were roughly 0.56, 0.68, and 0.82. I assigned trials to each of these three (6/13 for the ATCC_6919 and 12/15 and 8/12 for the two ATCC_11827). The fourth culture I checked had an OD of roughly 0.4, so I was hoping to add glycine on Monday.
7/14/2014
I was not able to get into the building over the weekend, so two of the four cultures had overgrown in that time. The OD of both of these were over 1.4. The two that remained were both ATCC_11827 that I had added glycine into, so I made competent cells from these and tried to perform an electroporation. The OD of 8/12 was approximately 1.05 and the OD of 12/15 was approximately 1.2. The machine sparked, but it did so between the machine’s metal and the metal on the electroporation cuvette. Does this constitute an arc? I took it to be an arc at the time, and so disposed of the cells and the cuvettes, but this may have been an error in retrospect. I saved several microcentrifuge tubes of each trial in the -80 freezer for later use.
We also discovered at the team meeting that Cheong had not actually GROWN the bacteria at different temperatures, but incubated them at that temperature for 5 hours after growing them, but before making competent cells from them. I may start trying this technique if the previous technique did not work.
Also, glancing at the plates for our previous electroporations, I could see that the plate with no erythromycin showed a lot of growth on it, with not much on the erythromycin plate. This shows that the cells were not killed during the competent cell preparation or the electroporation. I will look in more detail in the next few days to check for small colonies.
7/15/2014
Today I started 2 new liquid cultures in the 37 C room, and updated the lab notebook. I may perform electroporations from the frozen competent cells a little later, but no promises.
We only have 2 plates with erythromycin on them remaining, while 4 plates with no antibiotic are still available for use. Some of the erythromycin plates somehow got contaminated with bacteria, so they had to be thrown out. I will need to make some more of each of these plates in the next few days.
7/16/2014
Today I made 4 plates with erythromycin and checked the plates downstairs for growth. There did not seem to be any growth on the plates with erythromycin on them, but there was growth on all the plates without this resistance. This indicates that I did the experiment correctly (or at least mostly) and that the cells didn’t die during the procedure, but there were no transformants. Ching came to look with me, and said also that he didn’t see any transformed colonies.
I made a stock solution of approximately 1 mL of 50 mg/mL erythromycin in ethanol, then added 20 uL of this into a 100 mL batch of A media with agar. I also saved 100 mL of A media with no agar and no erythromycin in a bottle for later use. The four plates look fine and are now in the fridge, ready for use.
7/17/2014
Today, I checked the OD of the cultures I started on Tuesday. The were both approximately 1.2, much too high to add glycine, so I threw them out. These were both of strain ATCC_11827, and grew much faster than I would have expected.
We also discovered that Cheong’s protocol was actually slightly different from ours in that he grew p. acnes to stationary phase only at 37 degrees C, then aliquotted it (100 uL) into several 5 mL tubes of growth media. They then incubated it for 5 hours at multiple temperatures (for it to get into exponential phase, presumably), then made competent cells.
Carlos (who got back today) told me that this is essentially what we have been doing, unfortunately after I had already dumped the cultures we had growing upstairs because I was confused about the implications of this difference.
Ching suggested a few ideas to optimize our protocol. These ideas were: plating ALL of the cells after recovery instead of just a portion of them (do this by spinning down the cells, taking out most of the media, then resuspending the cells in less media and plating them. He also suggested varying the temperature of recovery, because a lower recovery temperature may inhibit the restriction enzymes in p. acnes.
7/18/2014
Today, I made 2 liquid cultures of p. acnes to grow in 24 C, because it will take longer, and so the weekend will give these cultures a head start. I also emptied all the anaerobic (Lock-n-Lock) chambers that I could and cleaned bottles that I needed to use for next week.
Stephen and I also discussed the project, and how to optimize our protocol, because we decided that the Cheong buffer was most likely not beneficial to the project (it contains salts, which leads to arcing). We changed this parameter to the incubation temperature of the cells during recovery.
Today, I did notice a single colony on one of the plates with erythromycin, which Carlos said could probably be p. acnes, although it would need to be verified. Even if it is p. acnes, he said, one transformation isn’t a high enough efficiency to actually mean anything.
<1--/div> <7/21/2014
The ATCC_11827 strain miraculously grew too much over the weekend while the other strain did not. I suspected not only p. acnes was present in the culture, so Carlos showed me how to check this by using the phage, and gave me some phage so I could check for this in the future. I am currently waiting for it to grow up (overnight) to see if there was contamination. This was the same strain that grew too quickly last week, so I have reasons for my suspicions.
I also discovered that one of our bottles of media had been contaminated, so I disposed of it and will probably have to make some more later (there are only 160 mL remaining).
I started 2 new liquid cultures of ATCC_6919 to incubate in 37 C. Hopefully they will grow quickly. I also measured the OD of the not-overgrown ATCC_11827 culture. It was 0.155, so too early to add glycine.
7/22/2014
I added glycine to the ATCC_6919 that was growing upstairs, because it was definitely ready, just by sight (hopefully it is p. acnes). It will be run 11/16 on the new chart (version 2), and I plan to make competent cells from it tomorrow. I also checked the plate downstairs with the phage added into it and there was definitely another type of bacteria than just p. acnes growing, and the phage hadn’t disposed of it. Today, we are going to do the same experiment for the overgrown culture of ATCC_11827.
Today I also made more media (3 bottles of 60 mL without agar, and 1 bottle of 120 mL with agar).
7/23/2014
Today I made competent cells from the 11/16 culture. The OD was 0.938. I will also try an electroporation with these comp. cells and will plate them tomorrow using the modifications. The parameters are: 0.6 lysozyme (which I made this morning), 2.00 ug DNA, 15 kV/cm electric field, 37 C post-zap temperature, and no TypeOne present.
I added glycine into the 2 cultures that were growing in 37 C. The two OD’s were approximately 0.51 and 0.42. Tomorrow or the next day, I plan to make comp. cells from these. They will be runs 15/3 and 3/11, respectively.
7/24/2014
Plated the electroporated cells from yesterday.
I also checked the OD of the two other cultures, which came out to be 0.819 and 0.816. I then made comp cells from these two cultures, which (again) are runs 15/3 and 3/11. I then electroporated one round of these comp. cells.
We also discussed how to check if the colony that was on the previous plate (run 1/8 from the old list) was actually p. acnes and if it was actually transformed. I will need to grow it up on a plate with erythromycin (or in a liquid culture) anaerobically, then streak it aerobically to make sure it’s p. acnes, and perform some PCR to verify that the vector is in there.
7/25/2014
Plated the electroporated cells from yesterday.
Discussed with Carlos and Ching how to check the transformant, and started a small liquid culture (in erythromycin) as well as plating the cells on an erythromycin plate.
I then created two new liquid cultures of ATCC_6919 to incubate in 24 degrees.
7/28/2014
First thing in the morning, I checked all the plates and liquid cultures. There seemed to be some colonies on the run 15/3 plate, but the others I couldn’t see very well. The liquid culture with erythromycin in it (to check for the transformant) was definitely grown up, maybe too much so I suspect it has something to do with how it was kept, although it is also possible that the erythromycin didn’t make it all the way into the media. One of the two liquid cultures in 24 C seemed to have grown up enough to check the OD, so I’ll do that today, and will likely add glycine to it. Finally, the plate with the phage treatment on it was still not entirely grown up in my opinion, which is a bit strange. I’ll ask Carlos what he thinks.
The OD of the liquid culture was below 0.4, so I have decided to give it another day. Also, Carlos (or that entire side of the Hatfull Lab) is not here yet, so I will have to hold off on asking any questions.
Carlos arrived and checked the plate, saying that it is p. acnes because a section of the lawn was missing, right where the p. acnes phage was placed. I can now make cultures from strain ATCC_11827.
07/29/2014
Today, the OD of the cultures I started last Friday were 0.513 and 0.462, and so are ready to have glycine added to them. These will be trials 7/6 and 13/5, both of which have 1% glycine added into them. Also, these are the last competent cells I will have to make of strain ATCC_6919.
I plan to pour more plates today (with erythromycin added into them) from the agar that I saved from before. I will be adding 24 uL of erythromycin to the entire batch before pouring. The media hadn’t completely melted (or had resolidified), so much of it was wasted on a bad plate before 4 more plates were made correctly. Thus, I now have 7 plates with erythromycin on them with which to complete this project for the summer.
These are the competent cells I have in the -80:
(from old list): 2/1, 8/12, 1/8, 12/15, 14/9, 10/2
which translate to (on new list): 9/10, 5/9, 1/2, 15/3, 4/5, 6/13
Cells in -80 (new list):
11/16, 3/11, 15/3
07/30/2014
I checked the OD’s of the two cultures with glycine added. Trial 7/6 was 0.898 and trial 13/5 was 0.573. I decided to make comp. cells from trial 7/6, but not from 13/15, which I will leave until (probably) Friday.
I should also mention a change that I have been making to the protocol. The high level for electric field is now 12.5 volts/cm, because this is the highest that their electroporator can go with 2 mm cuvettes. I have used this level for all of the new trials.
07/31/2014
Today was Science Palooza, but I still was able to add glycine to the two cultures growing in 37 degrees. Their OD’s were 0.792 and 0.896, so I let them sit off of the shaker overnight as they grew with glycine.
I also checked the plate that corresponded to run 11/16, and the plate somehow contained a lawn. The other two trials will be officially checked tomorrow, but I can already see a decent amount of transformed colonies.
08/01/2014
Today, I made competent cells from all three cultures that had been growing with glycine (runs 10/8, 13/15, and 14/16), and saved them in the -80. This was after Stephen and I decided on a plan to move forward, which includes verifying the colonies on the plates we already have with transformed colonies, and then waiting to do the rest of the electroporations until the semester starts. I now have 9 different preparations of competent cells in the -80 that could be readily electroporated.
I discussed with Carlos how to check the “transformed colonies” that we have, and he explained that I would need to streak each of the colonies (multiple on one plate), and start a small liquid culture of one of the small colonies, and one of the big ones. He gave me the code to get in and do all of this tomorrow, before waiting all of next week. He also showed me how to easily count the colonies using their lab equipment, so yay. For the plate with the lawn, I need to restreak it on erythromycin plates and hope for single colonies.
PCR Reaction of Hsp60 Promoter June 25, 2014
Purpose: Amplification of Hsp60 Promoter for insertion into biobrick
Reaction Reagents:
1. mCherry Fushion hsp60 promoter template
2. Primers
a. Hsp60BBfwd
b. Hsp60rev comps
c. RBS_BBrev comps
3. PCR Buffer
4. Double distilled water
5. DNA Polymerase
6. 1kb DNA Ladder
Primer Numbering and Concentration:
1. Hsp60BBfwd: 29.1 nmol/100ul
2. Hsp60rev comps: 26.2 nmol/100ul
3. RBS_BBrev comps 27.7 nmol/100ul
Final Concentration 100 uM of DNA
Reagent Table 1:
Reagent |
3x (ul) |
Clear Color Buffer |
12 |
dNTP's |
1.2 |
Primers: 1 + 3 |
6 |
Template DNA |
3 |
DNA Polymerase |
0.6 |
ddH2O |
37.2 |
Total Volume |
60 |
Reagent Table 2:
Reagent |
3x (ul) |
Clear Color Buffer |
12 |
dNTP's |
1.2 |
Primers: 1 + 2 |
6 |
Template DNA |
3 |
DNA Polymerase |
0.6 |
ddH2O |
37.2 |
Total Volume |
60 |
Loading Buffer Concentration Table:
x= total loading volume
Reaction loaded (ul) |
Loading Buffer (ul) |
4 |
1 |
5 |
1.25 |
8 |
2 |
10 |
2.5 |
Lane
Sample
Amount Loaded (ul)
1
1kb DNA Ladder
2
2
(1) Hsp60BBfwd
(3) RBS_BBrev
6.25
3
Control, no primers
6.25
4
Ladder
6.25
5
(1) Hsp60BBfwd
(2) Hsp60BBrev
6.25
6
Control, no primers
6.25
It seems that too much template DNA was used hence the bands did not travel far
Liquid Culture with DAM- E. coli in LB
Thursday, June 26, 2014
1. Set up 3 liquid cultures with 4 ml of LB each and 4 ul of Ampicillin (1:1000 Concentration)
2. Incubated in 37˚C shaker overnight
Mini-Prep DAM- Negative Liquid Culture
Friday, June 27, 2014
Buffer Preparation:
Resuspension Solution: add provided RNAse Al store at 4˚C for up to 6 months
Wash Solution: Add 35 ml of 96% EtOH to 20 ml wash solution. Store at room temperature (RT); we have 100% EtOH
1. Pipet 1.5 mL of culture into microcentrifuge tube and spin for 2 minutes
2. Resuspend pellet in 250 mL of Resuspension solution
3. Add 250 mL of Lysis solution and mix by inverting tube
4. Add 360 mL of neutralization and mix immediately by inversion 4-6 times
5. Centrifuge for 5 minutes to pellet cell debris
6. Transfer supernatant to spin column
7. Centrifuge for 1 minute and discard flow through
8. Add 500 mL of wash solution and centrifuge for 30-60 seconds and discard flow through
9. Repeat wash using 500 mL of wash solution
10. Transfer spin column to fresh 1.5 mL tubes and add 50 mL of elution buffer to center of column
11. Incubate at RT for 2 minutes centrifuge for 2 minutes
12. Store flow through (purified plasmid DNA) at -20 degree Celsius.
Result:
Tube |
Concentration (ng/uL) |
1 |
29.1 |
2 |
29.5 |
3 |
18.4 |
4 |
25.9 |
5 |
18.5 |
6 |
16.8 |
7 |
23.9 |
8 |
15.6 |
PCR of Hsp60 with and without RBS
Friday, June 27, 2014
Making a 1:13 dilution of our template DNA which is 140 ng/uL
Components |
Amount (ul) |
deionized water |
12 |
Template DNA |
1 |
Reagent Table 1 (w/o RBS):
Reagent |
3x (ul) |
Clear Color Buffer |
12 |
dNTP's |
1.2 |
Primers: 1 + 3 |
3 |
Template DNA |
3 |
DNA Polymerase |
0.6 |
ddH2O |
37.2 |
Total Volume |
57 |
Reagent Table 2 (w/ RBS):
Reagent |
3x (ul) |
Clear Color Buffer |
12 |
dNTP's |
1.2 |
Primers: 1 + 2 |
3 |
Template DNA |
3 |
DNA Polymerase |
0.6 |
ddH2O |
37.2 |
Total Volume |
57 |
Primer Numbering and Concentration:
1. Hsp60BBfwd: 29.1 nmol/100ul
2. Hsp60rev comps: 26.2 nmol/100ul
3. RBS_BBrev comps 27.7 nmol/100ul
Final Concentration 100 uM of DNA
1.25 uL of loading dye + 5 uL PCR reaction
2ul 1 kb DNA ladder
Started gel at 4:26 pm
Stopped gel at 4:55 pm
Results:
There were no PCR Products
PCR with Hsp60
Monday, June 30, 2014
Reagent Table 1 (w/o RBS):
Reagent |
2.5x (ul) |
Clear Color Buffer |
25 |
dNTP's |
2.5 |
Primers: 1 + 3 |
12 (6 each) |
Template DNA |
6 |
Phusion |
1.25 |
ddH2O |
77.5 |
Total Volume |
125 |
Reagent Table 2 (w/ RBS):
Reagent |
2.5x (ul) |
Clear Color Buffer |
12 |
dNTP's |
1.2 |
Primers: 1 + 2 |
12 (6 each) |
Template DNA |
6 |
Phusion |
1.25 |
ddH2O |
77.5 |
Total Volume |
125 |
PCR Tube Labeling:
1 = fwd primer + rev primer RBS
2 = fwd primer + rev primer Hsp60 (without RBS)
New PCR Condition:
Time |
|
98 |
30 secs |
98 |
10 secs |
64 |
15 secs |
72 |
15 secs |
72 |
5 mins |
4 |
hold |
Some housekeeping tasks
Monday, June 30, 2014
0.8% Agarose Gel:
25 mL 1x TBE buffer
0.2 g Agarose
1uL EtBr
100 mL LB and autoclaved:
1g tryptone
0.5g yeast extract
1g NaCl
Loaded 1 kb of volume 1 ul and then 6.25 ul of samples (w/RBS in lane 2 and w/o RBS lane 3)
Gel started at 3:30 pm
Gel stopped at 4:40 pm
Made 10, 5 mL LB+AMP, E. coli dam- pBRES36a culture at 6 pm
Mini-Prep
Tuesday, July 1, 2014
Mini-prepped 50 mL of E. coli dam- pBRES36a in LB+AMP
Obtained:
450 ul of 17.6 ng/ul of DNA
50 ul of 14.6 ng/ul of DNA
Primer Walking and PCR Purification
Wednesday, July 2, 2014
Primer Walking:
Belle submitted the plasmid (pBRES36a) to GeneWiz for Primer Walking
PCR Purification:
Purified 45 mL of both RBS and no RBS PCR products using new SV Wizard Kit from Promega
Added 75 mL of 95% EtOH to membrane wash solution
Final Concentration:
Tube Labeling |
PCR Product |
Concentration (ng/uL) |
1 |
RBS |
17.0 |
2 |
No RBS |
20.9 |
Restriction Digest of PCR Product with XbaI and PstI
Thursday, July 3, 2014
Result
1. 8.6 ug/mL for RBS PCR product
2. 11.5 ug/mL for no RBS PCR product
Mini-Prep
Tuesday, July 8, 2014
Mini-prepped 5x5 mL of E. coli dam- pBRES36a in LB+amp
Ran out of lysis solution ,unable to mini prep the remaining 25 mL
Yield:
5x50 mL = 250 mL
Concentration: 42 ug/mL, equivalent to10.5 ng plasmid
Testing old Tubes and Preparing LB Agar (100mL)
Wednesday, July 9, 2014
Testing Old Mini-prep Columns and Tubes:
Use 20 uL of 3.0 ug/mL DNA
Result
New tubes: 52 ug/mL
Old Tubes: 20 ug/mL
Conclusion: old tubes can no longer be used purchased new tubes
100 mL LB Agar and autoclaved:
1g tryptone
0.5g yeast extract
1g NaCl
1.5 g Agar (1.5%)
100 mL Water (ddH2O)
50 mL aliquots
Ligating Hsp60 Inserts Into Vector Backbone
Monday, July 14, 2014
1. Digestion
Materials:
Reagent |
Amount (ul) |
DNA |
40 |
Buffer |
5 |
XbaI restriction enzyme |
1.5 |
PstI restriction enzyme |
1.5 |
ddH2O |
2 |
Total |
50 |
Procedures:
1. Mixed materials together in microcentrifuge tubes
2. Incubate 30 minutes at 37 degrees Celsius
2. PCR Purification (using Promega Wizard SV gel clean up kit)
3. Ligations
a. Ligation 1: RBS and Vector
b. Ligation 2: no RBS and Vector
Materials for Ligation 1:
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of RBS insert |
4 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
9.5 |
Materials for Ligation 2
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of no RBS insert |
3 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
10.5 |
Procedures:
1. 15 minutes at room temperature
2. Put on ice until transformation
4. Transformation (see transformation protocol)
Housekeeping
Monday, July 14, 2014
30 mL LB Agar and autoclaved:
0.3 g tryptone
0.18 g yeast extract
0.3 g NaCl
0.45 g Agar (1.5%)
30 mL Water (ddH2O)
Chloramphenicol (CAM)
10 mg in 1 mL of 95% EtOH
5ug/mL workign concentration is needed
Used 35 ug/mL final
Prep 50 mL LB agar +CAM:
0.5 g tryptone
0.25 g yeast extract
0.5 g NaCl
0.75 g Agar (1.5%)
48 mL Water (ddH2O)
25 uL of 10 mg/mL CAM
Competent cells growing
Housekeeping and Ligation
Wednesday, July 16, 2014
Prep 75 m
0.75 g tryptone
0.375 g yeast extract
0.5 g NaCl
1.125 g Agar (1.5%)
50 mL Water (ddH2O)
Ligations
Ligation 1: RBS and Vector
Ligation 2: no RBS and Vector
Materials for Ligation 1:
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of RBS insert |
4 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
9.5 |
Materials for Ligation 2
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of no RBS insert |
3 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
10.5 |
Transformation:
50 mL comp cells + 5 uL plasmid (shipment plasmid + RBS/no RBS)
On ice for 2 minutes
Heat shock 42 degree Celsius for 45 seconds
Ice 2 minutes
550 mL SOC media into mixture
Shake at 37 degree Celsius for 1 hour (recovery)
Plate onto CAM plate + incubate at 4 pm
Plan + CAM plate
Friday, July 18, 2014
Plan of Attack with pBRES36a
1. Digest with individual restriction enzymes and a negative control
a. No enzyme
b. XbaI
c. PstI
d. SpeI
e. EcoRI
2. Visualize on Gel
3. Double Digest with Pair from Step 1
4. Visualize on gel
5. Map the sequence
CAM plates made with 35 ug/mL
Transformation with:
1. Ligation product (2 of them)
2. Plasmid that's known to express CAM (for control)
7/20/14
2nd Double Digestion: For the purpose of confirming that there was no mix up in the previous digestion.
Components |
Amount (uL) |
10x Fast Digest Buffer |
2 |
pBRES36a (42ng/uL) |
16 |
SpeI |
1 |
PstI |
1 |
Total |
20 |
Lane # (from left) |
Sample |
1 |
Ladder (not visible) |
2 |
No enzyme |
3 |
PstI and SpeI |
4 |
No enzyme |
5 |
PstI and SpeI |
Housekeeping
Wednesday, July 23, 2014
Cam plates: 35 ug/mL, dilute 1:1000
Used 75 mL, used 75 ul
For 4 plates
Transformation with RBS:
50 mL E. coli comp cells, 5 uL RBS ligation
50 mL E. coli comp cells, 5 uL no RBS ligation
Transformed and recovered for 1 hour
Plated 50 uL of transformed cells onto cam plates
Restriction Digest of pBRES36a Plasmid
Monday, July 28, 2014
1. Digestion
Purpose: cut with restriction enzymes to provide a rough map for the pBRES36a plasmid and confirm the plasmid's identity
Materials:
Reagents |
Amount (ul) |
pBRES36a plasmid |
17 |
Buffer |
2 |
Restriction Enzyme |
1 |
Total Volume |
20 |
Procedures:
1. Mix reagents together and spin down gently.
2. Incubated for 10 minutes
3. Pipet up and down gently to mix samples
4. Load each sample onto the agarose gel
5. Run electrophoresis according to the conditions specified.
Enzyme Used:
1. XbaI
2. PstI
3. SpeI
4. EcoRI
2. Gel Electrophoresis
Run 1: Gel Set Up (7/18/14)
lane |
Reaction/Reagent |
Amount loaded (ul) |
1 |
1 kb Marker |
1 |
2 |
Plasmid + XbaI |
5 |
3 |
Plasmid + PstI |
5 |
4 |
Empty Lane |
0 |
5 |
Plasmid + SpeI |
5 |
6 |
Plasmid +EcoRI |
5 |
7 |
Plasmid only |
5 |
Run 1 Condition:
1. 2.0 hours
2. 80 Volts
3. 0.8% Agarose Gel
4. 1x TBE Buffer
Run 2: Gel Set Up (7/28/14)
lane |
Reaction/Reagent |
Amount loaded (ul) |
1 |
1 kb Marker |
1 |
2 |
Plasmid + XbaI |
5 |
3 |
Plasmid + PstI |
5 |
4 |
Plasmid + SpeI |
5 |
5 |
Plasmid +EcoRI |
5 |
6 |
Plasmid +SpeI and EcoRI |
5 |
7 |
Hsp60 + XbaI and PstI |
5 |
8 |
mCherry + XbaI and PstI |
5 |
Run 2 Condition:
1. 1.5 hours
2. 80 Volts
3. 0.8% Agarose Gel
4. 1x TBE Buffer
July 28, 2014 con’t:
Results
Run 1 (7/18/14) Left and Run 2 (7/28/14) Right
Ligation, Transformation, Selection
Thursday, July 24, 2014
Ligations
Ligation 1: RBS and Vector
Ligation 2: no RBS and Vector
Materials for Ligation 1:
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of RBS insert |
4 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
9.5 |
Materials for Ligation 2
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of no RBS insert |
3 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
10.5 |
Incubate at RT for 15 minutes
Transformation
1. 50 mL E. coli comp cells
2. 6 uL plasmid
3. 2 min on ice
4. 45s heat shock at 42 degrees Celsius
5. 2 min on ice
6. 950 mL SOC growth media
7. 1 hour recovery at 37 degrees Celsius
Selection:
plate 60 mL of transformed cells onto selection plate (LB + CAM plates)
8/1/14
1. Take out overnight liquid culture around 11:00 am
2. Mini prep iGEM plasmid parts
Part |
Amount (ng/uL) |
Desaturase |
42.6 |
Cathelicidin |
12.7 |
mCherry |
12.7 |
8/4/14 Lab
Make 1% Gel (30 mL):
0.3 g Agarose
30 mL 1x TBE
1 uL EtBr
Restriction Digests:
hsp60 PCR products:
RBS: no RBS:
6 uL ddH2O 6 uL ddH2O
2 uL Buffer 2 uL Buffer
1 uL XbaI 1 uL XbaI
1 uL PstI 1 uL PstI
10 uL PCR product 10 uL PCR product
iGEM Parts:
mCherry: Cathelicidin: deSaturase:
6 uL ddH2O 6 uL ddH2O 6 uL ddH2O
2 uL FD Green Buffer 2 uL FD Green Buffer 2 uL FD Green Buffer
1 uL XbaI 1 uL XbaI 1 uL XbaI
1 uL PstI 1 uL PstI 1 uL PstI
10 uL mCherry 10 uL Cathelicidin 10 uL deSaturase
PCR Purification:
Run PCR purification of hsp60 digests (RBS and no RBS)
1: RBS concentration-
2: no RBS concentration –
Gel Electrophoresis:
Ladder – mcherry – cath – desat - empty - cath – mcherry – desat
Failed. Will upload pic later
Ligation:
Ligate hsp60 RBS and no RBS into pSB1C3 backbones
RBS: no RBS:
9.5 uL ddH2O 10.5 uL ddH2O
3.5 uL backbone 3.5 uL backbone
4 uL insert 3 uL insert
2 uL buffer 2 uL buffer
1 uL ligase 1 uL ligase
Transformation:
Transform RBS and no RBS ligations into E. coli competent cells
5 uL plasmid
100 uL competent cells
Ice 2 minutes
Heat shock (42C) 45s
Ice 2 minutes
Add 950 uL outgrowth media
Recover with shaking (37C) for 1 hour
Plate onto selection plate (chloramphenicol)
Restriction Digest:
Cut mCherry prep, Cath. Prep., deSat. Prep, 1 RBS, 1 no RBS, 1 mCherry with XbaI and PstI
6 uL ddH2O
2 uL FD Green Buffer
1 uL XbaI
1 uL PstI
10 uL plasmid DNA
Gel Electrophoresis:
1 kb+ ladder – mCherry prep – Cath. Prep – deSat prep – 1 RBS – 1 no RBS – 1 mCherry -
8/5/15 Lab
Make 1% Gel (35 mL)
0.35 g Agarose
35 mL 1x TBE
1 uL EtBR
Restriction Digests:
Master Mix (for 6 reactions): x6.5 =
6 uL ddH2O 39 uL
2 uL Green Buffer 13 uL
1 uL XbaI 6.5 uL
1 uL PstI 6.5 uL
Combine 10 uL of Master Mix with 10 uL of each plasmid:
mCherry Prep, Cath. Prep, deSat prep., 1 RBS, 1 no RBS, 1 mCherry
Incubate 45 mins at 37C
Gel Electrophoresis:
Run 1 hour 30 mins
Lanes:
1 kb+ ladder – mCherry mini – Desat – Cath – 1 RBS – 1 no RBS – 1 mCherry – 1 kb+ ladder
08/08/14
Used the culture collected from 8/1/14 mini-prep for the subsequent restriction digest
1. Restriction digest (45 minutes)
2. Run agarose gel
Plasmid |
Expected Band (kb) |
|
1 |
Desaturase |
~1 |
2 |
||
3 |
Cathelicidin |
~0.1 |
4 |
||
5 |
RBS |
~0.3 |
6 |
||
7 |
No RBS |
~0.3 |
8 |
Digestion was incomplete and need to repeat experiment
Experiment was repeated on 8/9/14 and digestion was successful. The desire bands were excised and purified. Unfortunately the image file was lost due to a technical problem prior to routine file backup. However, further experiments continued to use parts isolated on this date and bands of correct size were present through all of these steps.
Lab Work 8/12/14:
Gel Purification:
Gel Fragment |
Gel + Tube Mass (g) |
Tube Mass (g) |
Gel Mass (g) |
Membrane Binding Solution Added (uL) |
DNA Concentration (ng/uL) |
1 Cath. Part |
1.133 |
1.050 |
0.083 |
83 |
2.9 |
2 Cath. Part |
1.146 |
1.050 |
0.096 |
96 |
3.9 |
3 Desat. Part |
1.173 |
1.050 |
0.123 |
123 |
3.1 |
4 Desat. Part |
1.192 |
1.050 |
0.142 |
142 |
3.1 |
1 Backbone |
1.160 |
1.050 |
0.110 |
110 |
3.2 |
2 Backbone |
1.152 |
1.050 |
0.102 |
102 |
3.2 |
3 Backbone |
1.151 |
1.050 |
0.101 |
101 |
3.2 |
4 Backbone |
1.200 |
1.050 |
0.150 |
150 |
4.3 |
5 Backbone |
1.201 |
1.050 |
0.151 |
151 |
6.6 |
|
|
|
|
|
N/A |
7 Backbone |
1.243 |
1.050 |
0.193 |
193 |
5.7 |
8 Backbone |
1.159 |
1.050 |
0.109 |
109 |
5.7 |
Backbone |
1.300 |
1.050 |
0.250 |
250 |
4.4 |
*Add 10 uL of Membrane Binding Solution per 10 mg of Gel slice
-Vortex Gel and solution
-Incubate at 55C for 10 mins to melt gel
-Spin Down
-Gel Purify
Miniprep:
Miniprepped mRFP1 and MelA liquid cultures
-used same elution buffer for two of the same sample to obtain double DNA concentration
Concentrations:
1 mRFP: 516 ng/uL
2 mRFP: 462.5 ng/uL
3 mRFP: 243.5 ng/uL
1 MelA: 117.9 ng/uL
2 MelA: 158.3 ng/uL
3 MelA: 146.1 ng/uL
Lab Work 8/13/14:
Restriction Digest:
Digesting 1 mRFP, 2 mRFP, 3 mRFP, 1 MelA, 2 MelA, 3 MelA with XbaI and PstI.
20 uL Reactions: Master Mix:
5 uL Plasmid DNA 13 uL FD Green Buffer
2 uL FD Green Buffer 6.5 uL XbaI
1 uL XbaI 6.5 uL PstI
1 uL PstI 78 uL ddH2O
12 uL ddH2O 104 uL Total
*Reactions are 5 uL Plasmid + 15 uL Master Mix
*There are 6 reactions. Master Mix is 6.5x
*Incubate for 45 minutes at 37C
Gel Electrophoresis:
Running a gel to check the validity of the mRFP1 and MelA mini-prepped DNA.
Expect to see mRFP1 band at 706 bp and MelA band at 1844 bp.
Lanes:
1 kb+ --- 1 mRFP --- 2 mRFP --- 3 mRFP --- 1 MelA --- 2 MelA --- 3 MelA --- EMPTY
Gel:
Gel Purification:
Gel Fragment |
Gel + Tube Mass (g) |
Tube Mass (g) |
Gel Mass (g) |
Membrane Binding Solution Added (uL) |
DNA Concentration (ng/uL) |
1 mRFP |
1.142 |
1.017 |
0.125 |
||
2 mRFP |
1.143 |
1.009 |
0.134 |
||
3 mRFP |
1.149 |
1.012 |
0.137 |
*Did not complete Gel Purification
Plan for Tomorrow:
1) Finish Gel Purification (Possibly Re-Run. I believe I cut wrong fragment)
2) Make chloramphenicol plates
3) Resuspend YF1 & FixJ, Blue Light Sensor (Plate 1, Well 10N; 1:10N)
4) Resuspend FixK2 Promoter (Plate 1, Well 19G; 1:19G)
5) Transform and Plate (YF1 & FixJ) and FixK2 Promoter
If able to obtain Kanamycin:
1) Make Kanamycin plates (2-3)
2) Transform and Plate mCherry Bomb (3.4 uL of plasmid left)
Lab Work 8/14/14:
Plan:
1) Re-Run mRFP1 on Gel and then Gel Purify
2) Make Chloramphenicol Plates
3) Dilute mCherry Bomb and nanodrop
3a) If enough DNA is present, run PCR (1.4 ng/uL only)
3b) If not enough DNA is present, make Kanamycin plates
4) Resuspend YF1 & FixJ, Blue Light Sensor (Plate 1, Well 10N; 1:10N)
5) Resuspend FixK2 Promoter (Plate 1, Well 19G; 1:19G)
6) Transform and Plate (YF1 & FixJ) and FixK2 Promoter (Chloramphenicol)
7) Transform and Plate mCherry Bomb plasmid (Kanamycin)
mRFP1 Gel Electrophoresis:
-Create 25 mL or 1% agarose gel and let solidify
-Load digested mRFP1 DNA from 8/13/14
Lanes:
1 kb+ Ladder --- Empty --- 1 mRFP --- empty --- 2 mRFP --- empty --- 3 mRFP --- empty
Dilute mCherry Bomb and nanodrop:
-1 uL mCherry Bomb into 9 uL ddH2O
-Concentration of 1.4 ng/uL
Make Plates:
*1 Kanamycin; 3 Chloramphenicol
-Kanamycin working concentration of 50 ng/mL
-CAM working concentration of 25 ng/mL
Resuspend DNA:
*Added 10 uL ddH2O to each part to resuspend
Transform:
Transformed Blue Light Sensor, Blue Light Promoter, and mCherry Bomb
*Plates put in 37C incubator at 4:16 PM
Gel Purification:
Gel Fragment |
Gel + Tube Mass (g) |
Tube Mass (g) |
Gel Mass (g) |
Membrane Binding Solution Added (uL) |
DNA Concentration (ng/uL) |
1 mRFP |
1.195 |
1.014 |
0.181 |
181 |
3.6 |
2 mRFP |
1.217 |
1.002 |
0.215 |
215 |
2.0 |
3 mRFP |
1.158 |
1.012 |
0.146 |
146 |
4.6 |
Lab Work 8/15/15:
Morning:
Took out agar plates of Blue Light Sensor, Blue Light Promoter, and mCherry Bomb
*Colonies were spotted on each Plate
Evening:
Made liquid cultures for Blue Light Sensor, Blue Light Promoter, and mCherry Bomb
*5 mL per liquid culture
*2 Liquid cultures per plasmid
*Added CAM to Blue Light Sensor and Blue Light Promoter cultures at working concentration of 25 ng/mL
*Added Kan to mCherry Bomb culture at working concentration of 50 ng/mL
Lab Work 8/16/14:
Miniprep:
-Miniprepped liquid cultures of Blue Light Promoter, Blue Light Sensor, and mCherry Bomb
Concentrations:
Blue Light Promoter (1:19G): 44.5 ng/uL
Blue Light Sensor (1:10N): 36.6 ng/uL
mCherry Bomb: 35 ng/uL
Lab Work 8/18/14:
PCR:
-PCRing hsp60 out of plasmid mCherry Bomb
Master Mix |
Primers and DNA |
PCR Reaction |
|||||
Reagent |
1x Rxn (uL) |
2.5x Rxn (uL) |
Primer |
RBS (uL) |
no RBS (uL) |
Reagent/DNA |
Volume (uL) |
Buffer |
10 |
25 |
Fwd Primer |
2.5 |
2.5 |
Master Mix |
11.5 |
dNTP |
1 |
2.5 |
Rev Primer |
2.5 |
2.5 |
Primers and DNA |
7.5 |
Phusion |
0.5 |
1.25 |
mCherry Bomb |
2.5 |
2.5 |
ddH2O |
31 |
Total |
11.5 |
28.75 |
Total |
7.5 |
7.5 |
Total |
50 |
*Two 20 uL PCR reactions of each (RBS and no RBS) were carried out
PCR conditions:
98C for 30s
98C for 10s
66C for 30s x30 cycles
72C for 30s
72C for 5 min
4C holding
Gel Electrophoresis:
Ran two PCR products with a ladder on a gel. There were no bands shown. PCR was unsuccessful
Lab Work 8/19/14:
Restriction Digest:
Digesting Blue Light Sensor and Blue Light Promoter
20 uL Reactions:
5 uL Plasmid DNA
2 uL FD Green Buffer
1 uL XbaI
1 uL PstI
12 uL ddH2O
*Incubate for 45 minutes at 37C
Gel Electrophoresis:
Running a gel to check the validity of the Blue Light Sensor (1796 bp), Blue Light Promoter (250 bp), and PCR products (~380 bp).
Lanes:
1 kb+ ladder --- RBS PCR --- no RBS PCR --- Blue Light Sensor --- Blue Light Sensor --- Blue Light Promoter --- Blue Light Promoter --- empty
9/12/14
Goal:
1. PCR purify Hsp60 with and without RBS
2. Digestion of purified product with XbaI and PstI
3. Gel purification of digestion products
a. Cut out slices and store at 4 ℃
Experiments:
Tubes Labeled by MJ
Tube label |
Content |
1, PCR purification product, no RBS |
1st elution: PCR purification product, no RBS |
2, PCR purification product, no RBS |
2nd elution: PCR purification product, no RBS |
1, PCR purification product, RBS |
1st elution: PCR purification product, RBS |
2, PCR purification product, RBS |
2nd elution: PCR purification product, RBS |
1. PCR purification
Step |
Hsp60 with RBS (uL) |
Components |
Hsp60 without RBS (uL) |
1 |
34.5 |
Membrane binding solution |
34.5 |
2 |
700 |
Membrane Wash solution |
700 |
3 |
500 |
Membrane Wash solution |
500 |
4 |
50 |
1st elution with Nuclease free ddH2O |
50 |
5 |
30 |
2nd elution with Nuclease free ddH2O |
30 |
2. Digestion of purified product
Components |
Amount for Hsp60 with RBS (uL) |
Amount for Hsp60 without RBS (uL) |
10x Fast Digest Green Buffer |
2 |
2 |
PCR Product of Hsp60 |
7 |
7 |
XbaI |
1 |
1 |
PstI |
1 |
1 |
ddH2O |
13 |
13 |
Total |
24 |
24 |
Incubated at 37 ℃ for 30 minutes
2 reactions each were digested for 1st elution of both with and without RBS; 1 reaction each for the 2nd elution for both with and without RBS
3. Gel Set Up:
Lane |
Sample |
Amount (uL) |
Expected band size (bp) |
1 |
1kb DNA Ladder |
2 |
None |
2 |
Elution 1, no RBS digested |
5 |
~ 300 bp |
3 |
Elution 1, no RBS digested |
5 |
|
4 |
Elution 2, no RBS digested |
5 |
|
5 |
1kb DNA Ladder |
2 |
None |
6 |
Elution 1, RBS digested |
5 |
~300 bp |
7 |
Elution 1, RBS digested |
5 |
|
8 |
Elution 2, RBS digested |
5 |
4. Mass of tube and tube plus sample
Tube label |
Sample (lane #) |
Mass of tube (g) |
Mass of Tube plus sample (g) |
1 |
2 |
1.012 |
1.1 |
2 |
3 |
1.005 |
1.098 |
3 |
4 |
1.018 |
1.121 |
4 |
6 |
1.01 |
1.105 |
5 |
7 |
1.02 |
1.113 |
6 |
8 |
1.003 |
1.094 |
Goals:
1) Digestion of Blue Light Parts w/ X+P
2) Run Parts on Gel
3) Gel Purify Blue Light Parts, hsp60 parts, vector backbone
4) Ligate hsp60 + backbone
Digestion:
9 uL ddH2O
2 uL Fast Digest Green Buffer
1 uL XbaI
1 uL PstI
7 uL Plasmid (Blue Light Sensor and Blue Light Promoter)
20 uL rxn
Incubate at 37 C for 30 min
Gel Electrophoresis:
Lanes:
1 kb+ ladder – Blue Sensor – Blue Sensor – Blue Promoter – Blue Promoter – empty
Image at 30 mins: Image at 1 hour, 15 mins:
*Lanes 4/5 extracted at 30 mins
Purification:
Tube Weight (g) |
Tube + Gel Weight (g) |
Gel Fragment Weight (g) |
Membrane Binding Solution (uL) |
|
Backbone |
1.021 |
1.183 |
0.162 |
162 |
Blue Promoter |
1.016 |
1.198 |
0.182 |
182 |
Backbone |
1.016 |
1.187 |
0.171 |
171 |
Blue Sensor |
1.004 |
1.126 |
0.122 |
122 |
no RBS |
0.088 |
88 |
||
no RBS |
0.093 |
93 |
||
RBS |
0.095 |
95 |
||
RBS |
0.093 |
93 |
Ligation:
RBS no RBS
1 uL ligase 1 uL ligase
2 uL Buffer 2 uL Buffer
4 uL Insert 3 uL Insert
3.5 uL Backbone 3.5 uL Backbone
9.5 uL ddH2O 10.5 uL ddH2O
20 uL 20 uL
9/22/14
Goals:
1. Digestion of desaturase, and mRFP-1
2. Purification of desaturase and mRFP-1
3. Ligation into the vector
4. Digestion of 7 uL of plasmid DNA
5. Purification
Experiment:
Cut mRPF-1 with EcoRI and XbaI
Desaturase with EcoRI and SpeI
Components mRFP-1 |
Amount (uL) |
Components desaturase |
10x Fast Digest Buffer |
2 |
10x Fast Digest Buffer |
plasmid |
7 |
plasmid |
EcoRI |
1 |
EcoRI |
XbaI |
1 |
SpeI |
ddH2O |
9 |
ddH2O |
Total |
19 |
Total |
Gel:
Lane |
1 |
2 |
3 |
4 |
Components |
1kb DNA Ladder |
Desaturase (EcoRI+SpeI) |
||
Amount (uL) |
2 |
5 |
5 |
5 |
Lane |
5 |
6 |
7 |
8 |
Components |
1kb DNA Ladder |
mRFP-1 (EcoRI+XbaI) |
||
Amount (uL) |
2 |
5 |
5 |
5 |
9/24/14
mRFP-1 and desaturase plasmids were previously collected and this is the resulting digestion (9/22/14) followed by gel purification of the previous experiments.
1. Gel purification: and resulting concentrations:
Tube label |
Part excised |
Mass (g) |
Concentration (ng/uL) |
1 |
Desaturase Vector Backbone |
Not used since this is the backbone |
|
2 |
|||
3 |
|||
4 |
Desaturase Part |
0.124 |
3.9 |
5 |
0.123 |
4.7 |
|
6 |
0.095 |
4.2 |
|
7 |
mRFP-1 linearized plasmid |
0.095 |
4.4 |
8 |
0.103 |
4.4 |
|
9 |
0.133 |
4.8 |
|
9/26/14
1. Ligation reaction BH
Components |
Amount (uL) |
Insert (desaturase) |
9 |
Vector Backbone (single cut mRFP-1) |
3.5 |
Ligase |
1 |
Buffer |
1.5 |
ddH2O |
5 |
Total |
20 |
Negative control: just VB (labeled A)
Ligation 1 (labeled B)
Ligation 2 (labeled C)
2. Transformation into E. coli competent cells BH
Incubation at 37 ℃ on LB + CAM from 2 pm
Note: the plate was examined once at 6 hours and another time at 8 hours— yielded no usable colonies
3. Mini-Prep (BH) of colonies culture grown by MJ
Labeling maintained the same (1-6)
Resuspended in 50 uL of elution buffer
Undetermined concentration
Ligation and Transformation were repeated 2 times yet the reaction never yielded colonies that survived in LB+CAM culture.
Lab Work 9/29/14:
Diagnostic Digest:
Hsp60(no RBS) – mRFP1 (~1090 bp) 1) X+P
Blue Promoter – mRFP1 (~1050 bp) 2) X+P, 3) S+P
Hsp60 (no RBS) – Blue Sensor (~2160 bp) 4) X+P
MelA (~1896 bp) 5) X+P
20 uL Rxns
9 uL ddH20
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
*Incubate for 30 min @ 37C
Gel Electrophoresis:
1 kb+ ladder – 1 – 2 – 3 – 5 – 4 – empty
Gel @ 30 mins Gel @ 1 hr, 30 mins
*Note: Band 3 was extracted after 30 minutes. The lower of band 5 was extracted at 1 hr, 30 min
Gel Extraction:
Blue Promoter – mRFP1 S+P gel weight: 0.086 g
Hsp60 (no RBS) – Blue Sensor X+P gel weight: 0.062 g
Made 6 CAM plates & 2 AMP plates
Transformation:
Transformed RBS (4:1N) and Terminator (4:16G).
Plate onto AMP plates
Lab Work 10/1/14:
Mini Prep:
Mini Prep RBS and Terminator Liquid Cultures
Gel Purify:
Add 86 uL Membrane Binding Solution to Blue Promoter – mRFP1 (S+P) part
Add 62 uL Membrane Binding Solution to hsp60 (no RBS) – Blue Sensor (X+P) part
Restriction Digests:
Terminator (E+X) (~2150 bp) Cathelicidin (E+S) (~123 bp)
RBS (E+X) (~2082 bp) Blue Promoter (E+S) (~250 bp)
20 uL rxns:
9 uL ddH2O
2 uL FD Green
1 uL EcoRI
1 uL XbaI/SpeI
7 uL Plasmid
Incubate 30 mins @ 37C
Gel Electrophoresis:
Lanes:
1 kb+ ladder – Terminator (E+X) – Cathelicidin (E+S) – RBS (E+X) – B. Promoter (E+S) – empty
*Run Gel 15 minutes
Gel Purifiy:
RBS (E+X)
Blue Promoter (E+S)
Gel Fragment |
Gel + Tube Mass (g) |
Tube mass (g) |
Gel Mass (g) |
Membrane Binding Solution (uL) |
DNA Concentration (ng/uL) |
|
|
|
|
|
|
|
|
|
|
|
|
RBS (E+X) |
1.033 |
0.985 |
0.048 |
48 |
1.7 |
Blue Promoter (E+S) |
1.082 |
1.005 |
0.077 |
77 |
1.8 |
Ligation:
Blue Promoter – RBS
15 uL Rxn:
3.5 uL Insert (Blue Promoter)
9 uL VB (RBS)
1.5 uL Buffer
1 uL Ligase
0 uL ddH2O
Transformation:
Transform Blue Promoter – RBS onto AMP plate
Lab Work 10/4/14:
Morning:
Made Liquid Cultures of Blue Promoter – RBS part in Turbo E.coli Comp Cells
Afternoon:
Mini Prep:
Mini Prepped Blue Promoter – RBS : ??? ng/uL
Restriction Digest:
Blue Promoter – RBS (X/P)
Blue Promoter – RBS (S/P)
Terminator (S/P)
20 uL rxns:
9 uL ddH2O
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
Gel Electrophoresis:
Lanes:
1) 1 kb+ ladder
2) Blue Promoter – RBS (X+P) (~262 bp)
3) Blue Promoter – RBS (S+P) (~2350 bp)
4) Terminator (S+P) (~2150 bp)
Gel Purify:
Gel Fragment |
Tube (g) |
Gel + Tube (g) |
Gel (g) |
Membrane Binding Solution (uL) |
Concentration (ng/uL) |
Blue Promoter-RBS (S/P) |
1.002 |
1.067 |
0.065 |
65 |
5.0 |
Terminator (S/P) |
1.020 |
1.093 |
0.073 |
73 |
2.1 |
Ligation:
1) Blue Promoter – mRFP1 (S/P) (~3000 bp) + Cathelicidin (X/P) (~120 bp)
2) Blue Promoter – RBS (S/P) (~2350 bp) + Cathelicidin (X/P) (~120 bp)
3) Terminator (S/P) (~2150 bp) + hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp)
4) Blue Promoter – mRFP1 (S/P) (~3000 bp) + hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp)
Ligation |
VB bp |
VB concentration (ng/uL) |
Insert bp |
Insert concentration (ng/uL) |
VB (uL) |
Insert (uL) |
1 |
3000 |
2.9 |
120 |
3.9 |
4.00 |
8.50 |
2 |
2350 |
5.0 |
120 |
3.9 |
3.50 |
9.00 |
3 |
2150 |
2.1 |
2160 |
4.9 |
3.00 |
9.50 |
4 |
3000 |
2.9 |
2160 |
4.9 |
3.50 |
9.00 |
1.5 uL Buffer
1 uL Ligase
Transformation:
Blue Promoter – mRFP1 – Cathelicidin (CAM)
Blue Promoter – RBS – Cathelicidin (AMP)
Terminator – hsp60 (no RBS) – Blue Sensor (AMP)
Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (CAM)
Lab Work 10/5/14:
Ligation:
Ligation 3: Terminator (S/P) + hsp60 (no RBS) – Blue Sensor (X/P)
1.5 uL Buffer
1 uL Ligase
6 uL VB (Terminator)
6.5 uL Insert (hsp60-Blue Sensor)
Transformation:
Transform 3: Terminator – hsp60 (no RBS) – Blue Sensor
AMP Plate
MiniPrep:
Mini 1: Blue Promoter – mRFP1 – Cathelicidin 61.5 ng/uL
Mini 2: Blue Promoter – RBS – Cathelicidin 85.9 ng/uL
Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor 81.0 ng/uL
Restriction Digest:
1) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (X/P)
2) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (S/P)
3) Mini 2: Blue Promoter – RBS – Cathelicidin (X/P)
4) Mini 2: Blue Promoter – RBS – Cathelicidin (S/P)
5) Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)
20 uL rxns:
9 uL ddH2O
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
Gel Electrophoresis:
Lanes:
1) 1 kb+ ladder
2) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (X/P) (~1000 bp) (Part)
3) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (S/P) (~3100 bp) (VB)
4) Mini 2: Blue Promoter – RBS – Cathelicidin (X/P) (~370 bp) (Part)
5) Mini 2: Blue Promoter – RBS – Cathelicidin (S/P) (~2470 bp) (VB)
6) Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)(~3160 bp) (Part)
*After 30 mins, hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp) added to lane 7. Ladder added lane 8.
Gel Purify:
Gel Fragment |
Tube (g) |
Gel + Tube (g) |
Gel (g) |
Membrane Binding Solution (uL) |
Concentration (ng/uL) |
Mini 1: B.P. - mRFP1 - Cath (S/P) |
1.013 |
1.072 |
0.059 |
59 |
2.6 |
no RBS - Blue Sensor (X/P) |
1.016 |
1.065 |
0.049 |
49 |
1.2 |
Ligation:
1.5 uL Ligation Buffer
1 uL Ligase
Lig 1: Lig 2:
9 uL Blue Promoter (Insert) (~200 bp) 6 uL Terminator (VB) (~2100 bp)
3.5 uL RBS (VB) (~2100 bp) 6.5 uL hsp60 (no RBS) – B. Sensor (Insert) (~2160)
Lig 3:
3.5 uL B. Prom – mRFP1 (VB) (~3100)
9 uL hsp60 (no RBS) – B. Sensor (Insert) (~2160 bp)
Transformation:
Transform 1: Blue Promoter – RBS onto AMP plate
Transform 2: Terminator – hsp60 (no RBS) – Blue Sensor onto AMP plate
Transform 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor onto CAM plate
Make Liquid Cultures:
Made 2 5mL Liquid cultures of LB + AMP, Blue Promoter – RBS
Lab Work 10/6/14:
Make Liquid Cultures:
(AMP) Transform 1: Blue Promoter – RBS
(AMP) Transform 2: Terminator – hsp60 (no RBS) – Blue Sensor
(CAM) Transform 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor
Mini prep:
Mini 1: Blue Promoter – RBS
Mini 2: Terminator – hsp60 (no RBS) – Blue Sensor
Mini 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor
Restriction Digest:
1) Blue Promoter – RBS (X/P)
2) Blue Promoter – RBS (S/P)
3) Terminator – hsp60 (no RBS) – Blue Sensor (X/P)
4) Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)
20 uL Rxns
9 uL ddH2O
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
Gel Electrophoresis:
Lanes:
1) 1 kb+ ladder
2) Blue Promoter – RBS (X/P) (~262 bp)
3) Blue Promoter – RBS (S/P) (~2341)
4) Terminator – hsp60 (no RBS) – Blue Sensor (X/P) (~2236)
5) Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P) (~3100)
*Note: In lane four, the VB is ~2100, will need long separation to see. Part will be bigger than backbone
*Stop at 15 mins and check lane 2, if correct band is present, extract lane 3 before continuing.
*Run for another hour and fifteen before stopping a second time and extracting lane 4
Gel Purify:
Ligation:
Ligation 1: Blue Promoter – RBS (X/P) + Cathelicidin (S/P)
Ligation 2: Blue Promoter – mRFP1 – Cathelicidin (S/P) + Terminator – hsp60 (no RBS) – Blue Sensor (X/P)
Ligation |
VB bp |
VB Concentration (ng/uL) |
Insert bp |
Insert Concentration (ng/uL) |
VB (uL) |
Insert (uL) |
|
|
|
|
|
|
|
2 |
2500 |
2236 |
