Team:Michigan/Experiments
Experiments
Building pThermolyze*:
Four slightly different versions of pThermolyze were synthesized by Integrated DNA Technologies. Each version was synthesized from three linear parts. Part A had the TlpA promoter followed by T4 antiholin and the TlpA36 repressor protein, flanked by NheI and BglII restriction sites. Part B included ampicillin resistance gene and placed the plasmid’s origin of replication between BglII and HindIII restriction sites. There were four variations of Part C, each of which had T4 holin and T4 endolysin, but with a different constitutive promoter in front of them (or no promoter in one case). All four Part Cs were flanked by HindIII and NheI restriction sites.
Once the parts were synthesized, we performed the appropriate restriction digests on each part using NEB restriction enzymes. We then used a Qiagen PCR cleanup kit on each part before ligating them together using NEB T4 ligase. After ligating at room temperature for 48 hours, the reactions were transformed into NEB DH5a chemically competent E. coli cells. These transformations were plated on Amp-LB plates and grown overnight. 5mL liquid LB-Amp cultures were prepared from colonies picked from the plates the next day and themselves grown overnight. DNA was extracted from these liquid cultures using a Qiagen miniprep kit.
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*Initially our plan was to use gibson assembly, followed by several rounds of restriction digests and ligations to assemble our plasmid. Despite multiple attempts (see lab notebook), we decided to switch to the method described above.
Testing pThermolyze:
Experiment #1: How do different promoters in pThermolyze affect cell growth at 37C and 25C?
For each of our four pThermolyze constructs (three promoters -T, L, and J- on part C and one with no promoter), two 50mL liquid LB-Amp cultures of E. coli transformed with our construct were grown in a shaking incubator at 37C until they had reached an OD of between 0.2 and 0.4 (read using a spectrophotometer at A540). The same was done for E. coli containing the pUC19 plasmid. When all ten of these cultures had OD between 0.2 and 0.4, initial readings at A540 were recorded. One of each of the five types of cultures (pUC19, no promoter, and promoters T, L, and J) was then shifted to a 25C shaking incubator. Every 20 minutes for 6 hours (or until a cultures OD went above the measurable limit of 2.5), A540 was measured and recorded.
This experiment was performed three times.
Experiment #2: Can pThermolyze transformed cells be stored as glycerol stocks and successfully recovered?
Glycerol stocks were produced from liquid cultures of E. coli transformed with each of our four pThermolyze constructs (three with different promoters -T, L, and J- on part C and one with no promoter on part C) and from a liquid culture of pUC19 transformed cells. After the glycerol stocks were stored for three days at -80C, prewarmed (37C) LB-Amp liquid cultures were inoculated with the glycerol stocks. These cultures were allowed to grow overnight in a 37C shaking incubator. 200uL of each culture was plated on LB-Amp plates and grown overnight in a 37C incubator. Plates were counted the next day.
Experiment #3: Can pThermolyze transformed cells be recovered from liquid cultures after storing at room temperature for 4 hours?
Liquid cultures of E. coli transformed with each of our four pThermolyze constructs (three with different promoters -T, L, and J- on part C and one with no promoter on part C) and a liquid culture of pUC19 transformed cells were each separated into five 1mL aliquots. 200uL of 1 of one aliquot of each type was plated on LB-Amp plates and incubated at 37C. Every hour, another 200uL of a new aliquot of each type was plated and incubated at 37C. After 24 hours since it was plated, each plate was examined and colonies were counted.
Experiment #4: Can pThermolyze transformed cells be recovered from plates after storing at 4C for 16 hours?
200uL of liquid cultures of E. coli transformed with each of our four pThermolyze constructs (three with different promoters -T, L, and J- on part C and one with no promoter on part C) and a liquid culture of pUC19 transformed cells were each plated on LB-Amp plates. These plates were stored at 4C. After 16 hours, 3 colonies were picked from each plate and used to start 5mL LB-Amp liquid cultures. These cultures were grown overnight at 37C and inspected for growth.
Protocols
Experiment 1:
Setup:
Start with 5 types of small liquid cultures grown overnight shaking at 37C: pUC19 (positive control), pThermolyze with part C without promoter (negative control), pThermolyze with part C and promoter J, pThermolyze with part C and promoter T, pThermolyze with part C and promoter L.
Set shaking incubators at 37C and 25C respectively.
1. Add 50mL prewarmed (37C) LB-Amp to 10 250mL flasks. Cover each with tin foil with a hole poked through for oxygenation.
2. Add 1.5mL from each 5mL starter culture to two 50mL cultures (ie: 1.5mL from the pUC19 starter culture into one 250mL flask and another 1.5mL into another 250mL flask).
3. Grow all 10 50mL cultures at 37C until A540 is between 0.2 and 0.4. Return sample to culture after measuring each sample.
4. When all cultures are between 0.2 and 0.4, record A540 for each culture (Time 0) and move one of each pair (P, C, etc) to the 25C shaker.
5. Every 20 minutes take measurements of A540 for all cultures. Return sample to culture after measuring each sample. Stop at 6 hour mark.
Agarose Gel Electrophoresis:
1. Fill an erlenmeyer flask with 50ml of 1X TAE buffer.
2. Weigh out and add 0.75g of agarose to make 1.5% gel. Mix by swirling the flask
3. Microwave for ~2.5 minutes without boiling. Then run the beaker under cold water until the glass has cooled.
4. Add 5uL of GelRed and mix gently by swirling.
5. Pour the beaker’s contents into the gel mold.
6. Add plastic “combs” to the mold to make wells.
7. Push any large bubbles to the side walls if possible using a spare comb or pipet tip.
8. Let the gel cool and solidify (~10-20 min).
9. Once the gel is solid, move it into the electrophoresis device. Make sure the ‘top’ end of the gel (with the wells) is on the same side as the negative terminal.
10. Pour 1X TAE buffer to the fill line.
11. Slowly and gently remove the plastic combs from the wells.
12. Select a ladder to use based on what kind of samples you’re running.
13. Load 10uL of the ladder into the leftmost well.
14. For each sample, in a small PCR tube, add 10uL of the DNA sample and 2uL of 6X purple loading dye and mix by pipetting up and down.
15. Load each sample into a well in the same way you loaded the ladder.
16. Run the gel.
Transforming DH5-Alpha strain E. coli Cells:
1. Warm the water bath to 42C and set shaker to 37C.
2. Thaw the plasmid DNA on ice.
3. Thaw one 50 uL vial of DH5-Alpha cells on ice.
4. Pipet 1-5 uL of the plasmid into the vial of cells, mix by gently tapping side of tube.
5. Store the extra plasmid at -20 C.
6. Put the tube of cells and DNA in ice for 30 minutes.
7. Make sure the hot water bath is at 42 C.
8. Put the vial in the 42 C water bath for exactly 30 seconds. Do not mix or shake.
9. Immediately put the vial back on ice for 5 min.
10. Add 950 uL of room temperature SOC medium to the vial.
11. Tape the tube of cells and DNA to a shaking incubator, and shake/incubate at 37 C and 225 rpm for exactly an hour.
12. Transfer 10 uL and 500 uL of the culture onto two petri dishes with correct antibiotic for the plasmid.
13. Spread evenly on plates using disposable sterile spreader.
14. Incubate the plates overnight (~12-20 hrs) at 37 C.
Miniprep:
We use Quiaprep spin kits for our minipreps.
1. Transfer 1.5 ml of the bacterial culture to a 1.5 ml eppendorf tube and centrifuge for 5 min at 6,000 rpm. Dump out supernatant from the eppendorf tube and add an additional 1.5 ml of culture. Repeat until your whole culture has been pelleted.
2. Pour out the supernatant into a liquid waste container.
3. Add 250uL of Buffer P1 to the tube containing the pellet, then pipet up and down until the pellet is completely resuspended. When you can no longer see any trace of the pellet, expel all liquid from the pipet back into the tube and proceed.
4. Add 250uL of Buffer P2.
5. With the lid closed flip the tube 4-6 times to mix. Wait 1 minute before proceeding.
6. Add 350uL of Buffer N3, then mix again, flipping it 4-6 times.
7. Centrifuge for 10 minutes at 13,000 rpm.
8. The kit comes with its own blue open top tubes with filters in them. Get one of these. There are two parts, the inner part with a filter and the outer part that’s just a tube. Label both of these parts to avoid any mix ups.
9. Transfer as much of the supernatant as you can (usually between 800uL and 1000uL) from the tube you just centrifuged onto the filter of the tube from the kit
10. Centrifuge the new tube with the filter in it for 60 seconds at 13,000 rpm.
11. Detach the bottom/outer part of the tube, dump its contents but keep the tube itself.
12. Put the inner part with the filter back inside the now empty outer part. Drip 500uL of Buffer PB onto the filter.
13. Centrifuge for 60 seconds at 13,000 rpm.
14. Detach the outer part and discard the flow-through again, then put the inner part back inside.
15. Drip 750uL of Buffer PE into the filter, again taking care not to touch the filter.
16. Centrifuge for 60 seconds. Discard the flow-through.
17. Centrifuge again for 60 seconds.
18. Discard the whole outer part of the tube, placing the inner part in a new, clean, and labeled eppendorf tube.
19. Drip 50uL of Buffer EB onto the filter, again taking care not to touch the filter.
20. Let it stand for 1-2 minutes.
21. Centrifuge for 60 seconds.
22. Throw out the inner portion of the tube.
23. Store the DNA in the freezer.
Restriction digest:
1. In a PCR tube, add 1ug of the DNA you want to digest.
2. Add 5uL of the 10X NEB Buffer you selected to match the restriction enzyme you’re using.
3. Add an amount of ddH2O which will give you 50ul final volume for your reaction.
4. Add 1ul of your chosen restriction enzyme. Stir the liquid with the pipette tip to make sure everything is fully mixed.
5. Incubate for 1 hour at the temperature specified by the restriction enzyme you’re using.
6. If the restriction enzyme you’re using is heat innactivatable then heat your reaction at the recommended temperature for 20 minutes.
7. If your enzyme is not heat innactivatable, you must use a PCR cleanup kit to remove your restriction enzyme.
Ligation:
1. Add 2uL of T4 DNA Ligase Buffer to a microcentrifuge tube.
2. Add 50ng of the vector DNA to the tube.
3. Add 37.5ng of the insert DNA to the tube.
4. Add nuclease free water to the tube so that the total volume is 19uL.
5. Add 1uL of T4 DNA Ligase. Gently mix the reaction by stirring with the pipette tip.
6. Incubate at 16C overnight or 1 hr at room temperature.
7. Put in 65C hot water bath for 10 minutes.
8. Chill on ice, then either transform into cells or store at -20C.
PCR:
1.Determine the number of PCR reactions you will be performing, then add 2.
2.Multiply all the following amounts by this new number to create the “master mix” for the PCR. Round up to the nearest uL. In a microcentrifuge tube add:
a) 25uL Q5 2X high fidelity master mix (NEB)
b)0.25uL 100uM forward primer
c)0.25uL 100uM reverse primer
d) 23.75uL Nuclease Free Water
3. Mix the master mix by pipetting up and down gently.
4. In small PCR tubes, on ice, add 2 uL (from 1-10 ng/ul stock for a 5kb plasmid) of the template DNA from each sample.
5. Add 48uL of the master mix to each PCR tube.
6. Load the PCR tubes onto the heat cycler.
7. Heat cycle through the program designed for your primers.
Gibson Assembly
1. In a PCR tube, add the following:
a) Xul of each of your gibson compatible fragments
b) 10uL 2X gibson assembly master mix
c) ddH2O so that the total volume is 20uL
2. In a second PCR tube, add the following:
a) 10ul of Positive Control mix
b) 10uL 2X gibson assembly master mix
3. Incubate the two tubes in the thermocycler at 50C for 15 minutes, then either store in the -20C freezer or immediately transform into NEB 5-alpha cells
Gel Imaging
1. Put blue plastic test plate on the imaging tray. Put the gel on the middle of the black part.
2. Move camera cone and cover the imaging tray. Cover completely.
3. Turn on the UV bulb.
4. Touch the “live” button on the computer screen. You should see the fake gel lines glow.
5. Turn bulb off.
6. Remove camera cone, replace the fake gel with your real gel.
7. Replace the camera cone.
8. Turn bulb on.
9. Keep adjusting camera until you can see the gel clearly.
10. Touch the “snap” button to capture your image.
11. Touch the “save button.”
12. Turn the bulb off, turn the imager off, wipe down the imaging tray and toss your gel.
Making LB Media
1. Get a glass screwtop container about twice the size of the amount of media you want to make.
2. Add the same amount of deionized water as you want media
3. Weigh out 25g of LB broth powder per 1L of media you want to make and add it to the flask.
- 2.5g for 100mL
- 5g for 200mL
- 6.25g for 250mL
- 12.5g for 500mL
5. Partially screw on the top and put a piece of autoclave tape on it.
6. Autoclave on appropriate liquid setting for the amount of liquid you have.
7. Let cool until at room temperature
8. If you are using the media immediately, you must wait until it has cooled to a temperature where you can comfortably touch the container before adding antibiotic.
9. Store at room temp with screw top completely closed.
Making LB Plates
1. Each plate takes 20-25mL of media.
2. Get a glass flask about twice the size of the amount of media you want to make.
3. Add the same amount of deionized water as you want media.
4. Weigh out 32g of LB agar powder per 1L of agar you want to make and add it to the flask.
- 3.2g for 100mL
- 8g for 250mL
- 16g for 500mL
6. Cover top in tin foil and put a piece of autoclave tape on it.
7. Autoclave on liquid setting.
8. Let cool until you can touch glass.
9. Add proper amount of antibiotic.
10. Pour evenly into plates. Once you pour into a plate, cover it ¾ of the way with its lid.
11. Let the plates solidify. Once they are solid, put the lids on all the way, and store them upside down in the fridge.
NEB Gel Extraction
1. Use a razor blade to cut the band you want out of the gel. Minimize the exposure of the gel to UV light since extra exposure damages the DNA.
2. Weigh the gel slice, then put the gel slice into a microcentrifuge tube.
3. Add 4uL of the Gel Dissolving Buffer per 1mg of the gel.
4. Incubate the tube in a 50C water bath, vortexing it occasionally until the gel is completely dissolved.
5. Get an open top tube with a filter in it.
6. Put the filter inside the tube, the add the now dissolved gel solution to the filter.
7. Spin in the microcentrifuge at 13,000 rpm for 1 minute.
8. Detach the filter part and discard the contents of the outer part of the tube.
9. Put the filter part back in the outer tube, then add 200uL DNA Wash Buffer.
10. Spin for another minute at 13,000 rpm.
11. Discard the contents of the outer tube again.
12. Spin again for a minute at 13,000 rpm.
13. Discard the contents of the outer tube again.
14. Take the inner filter part out and put it in a new, clean, microcentrifuge tube.
15. Label the new tube with as much detail as possible, this is the final storage tube for your DNA.
16. Add 10uL of Elution Buffer to the filter.
17. Wait one minute.
18. Spin at 13,000 rpm for a minute.
19. Take the filter out and discard it. The liquid in the microcentrifuge tube is your purified DNA.
