Difference between revisions of "Team:BostonU/Notebook/Protocols/Bacterial"

Revision as of 19:23, 18 September 2015

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Bacterial Protocols

To clone split protein constructs into our conditional dimerization backbones, we used the following protocols in our experimental pipeline.

1. We prepared LB broth and LB agar plates to grow E. coli K12 bacteria, the chassis for our cloning constructs.
2. We used the LB broth to grow up overnight bacterial cultures from frozen stocks - these bacteria contained relevant backbones and inserts that were previously created by our mentor Ben Weinberg.
3. We used an Epoch kit to Miniprep purified plasmid DNA from our overnight cultures, and subsequently used a Nanodrop to confirm DNA concentration.
4. We used restriction enzymes to digest backbone plasmid DNA.
5. We then performed PCR to amplify split inserts from insert plasmids - we designed primers to separately PCR N- and C- terminal domains of our proteins, corresponding to various split locations.
6. We gel purified both our backbone digestions and insert PCRs using agarose gel electrophoresis and an Epoch kit.
7. We used compatible restriction enzymes to digest insert DNA.
8. We PCR-purified our insert digestions using an Epoch kit.
9. We ligated together our compatibly-digested inserts and backbones to form newly cloned plasmids.
10. We transformed these ligation products into compatible E. coli K12 bacteria using heat shock. We plated our transformations onto LB agar plates overnight.
11. We picked colonies from our transformation plates and grew these up. We again used the Epoch Miniprep kit to purify these plasmids.
12. We performed test cuts and sequence verification to confirm that our plasmids contained the expected inserts.
13. We created frozen cell stocks of bacteria containing sequence verified plasmids in order to harvest more purified DNA for mammalian transient transfection experiments.

Making LB Broth and LB Agar Plates

Autoclaving Broth and Agar Solutions

1. Add 18g LB Broth Mix (for 900 mL LB broth) to deionized water; measured with weighboats
2. Add 40g LB Agar Mix (for 1L LB Agar) to deionized water; measured w/weighboats
3. Mix contents of broth or agar until powder is dissolved
4. Placed in autoclave with autoclave tape on top, labeled each, loosened cap, run on liquid cycle at 120 C for 1 hour
5. Removed autoclaved solutions with heat gloves and set on counter to cool
6. Plating the agar
7. Start flame with bunsen burner
8. Layout plates flat on the table with the lids on
9. Use carbenicillin antibiotic mix 1000x; (added 600 microliter to each agar which was 600mL LB agar solution)
10. Mix agar solution
11. Using electronic pipette, add 20 mL agar to each plate, opening lid slightly, moving plate to cool
12. Switch pipettes after every 8 plates
13. Dispose of pipettes in their wrapper into sharps container
14. Aliquot 50mL LB media tubes - purpose: so that when using media, in the case of contamination, only the 50mL volume will be contaminated rather than the entire stock solution
15. Open a flame
16. Use new pipette tip for each 50 mL tube
17. Label tubes
18. Resterilized opening and cap of stock LB broth before closing again

Growing Up Cultures

Inncoluate Plasmids from Frozen Stock

1. Have plasmids frozen at -80 C
2. Add 2 mL of LB media to a centrifuge tube
3. Prepare an ice tray or ice block for the frozen stocks to be held
4. Open flame
5. Scrape pipette tip on frozen cell stock and release the pipette tip into the appropriately labeled centrifuge tube of media
6. Immediately return frozen stocks to -80 C
7. Incubate plasmids in media overnight in 37 C 300rmp spinning incubator
8. Cell Stock Creation from frozen plasmids
9. All near an open flame
10. Label cryotubes for each bacterial cell stock
11. Took centrifuge tubes with grown up bacteria out of the incubator from yesterday (noting that all of the cells grew because the harvests were cloudy not clear)
12. Add 300 microliters of 10x glycerol to each cryo tube
13. Add 700 microliters of the bacteria cell stock into the appropriate cryo tubes
14. Store at -80 C

Miniprepping and Nanodropping

Miniprep

1. This process separates the plasmid DNA from the bacterial DNA so that we can use the plasmid DNA for our research.
2. Prepare the buffers according to the Miniprep Kit protocols that come with the Miniprep Kits.
3. Label the prepared buffers with the date that they were prepared and the initials of the researcher who prepared them.
4. Spin down the remaining bacterial cell stocks in the centrifuge tubes for 10 minutes at 3500 rpm.
5. Label microcentrifuge tubes while you wait: two microcentrifuge tubes, one intermediate and one final, for each bacterial cell stock
6. Prepare supernatant waste container by adding 10% bleach to an empty beaker
7. Pour supernatant from centrifuged tubes into supernatant waste container, so that only the pellet in the tube
8. Add 200 microliters MX1 buffer to each pellet
9. Resuspend the pellet by pipetting up and down
10. Add 250 microliters MX2 (this lyses the cells)
11. Flick bottom of tube to mix solution
12. Add 350 microliters MX3 within five minutes of adding MX2 (this neutralizes effects of MX2)
13. Flick bottom of tube to mix solution
14. Pipette about 900 microliters of pellet and solution into the labeled intermediate microcentrifuge tubes
15. Tabletop centrifuge all tubes at 1500 rpm for 10 minutes
16. Label Miniprep columns from Miniprep Kit
17. Insert Miniprep columns from Miniprep Kit into vacuum mantel
18. Pipette 850 microliters of supernatant from the microcentrifuge tubes into the center of the columns without touching the mesh, while making sure to get no cell debris
19. Add 500 microliters of WN into columns in columns
20. Let all of the WN pass through the mesh before adding WS
21. Add 700 microliters of WS into columns
22. Wait until all of the WS is gone before pulling out the columns
23. Turn off the vacuum
24. Insert the columns into their respective, labeled column holders
25. Dry spin the columns on tabletop centrifuge to get rid of the excess WN and WS at 1500 rpm for 2 minutes (so that the mesh now holds the DNA)
26. Insert column into microcentrifuge tube labeled final
27. Pour excess WN and WS from the column holder into the supernatant waste container.
28. Dispose of the column holders into the biohazard waste
29. Add 30-50 microliters of EB into the center of the column, which is in the final labeled microcentrifuge tube, making sure not to touch the mesh
30. Spin down final microcentrifuge tubes with the columns in them at 1500 rpm for 1 minute
31. Dispose of the columns into the biohazard waste

Using the Nanodrop Fluorospectrometer

1. Wipe down surface of the laser and the detector
2. Clear settings with 1 microliter water (the result should yield NaN)
3. Blank the settings with 1 microliter EB (the results should yield 0)
4. Measure all DNA concentrations using 1 microliter of DNA diluted in EB

Digesting Backbones

1. This process cuts the plasmid where the restriction sites are and then linearizes the backbone- we will purify the digestion later in order to separate the DNA fragment that was cut out that we do not want from the DNA backbone that we do want. To be used for Ligation.
2. *keep the enzymes cold!*
3. Lookup optimal buffers for restriction enzymes on NEB
4. Digest the pS#odd backbones with MluI and BspEI and a 3.1 buffer
5. Digest the pS#even backbones with EcoRI and KpnI and a cutsmart buffer
6. Check which buffers are optimal
7. Check the timing and temperature required for digestion
8. Check the inactivation temperature
9. when using multiple enzymes in the same tube, use buffers that have optimal activity for both
10. Digest 3-4 micrograms DNA for a complete digest (there’s also a test digest, using less DNA)
11. The total volume of the solution should be 50 microliters
12. To make master mix multiply below ratios by 7 for 6 digestions (1 extra just in case)
13. *keep enzymes on ice to not risk inactivation
14. 30 microliters deionized autoclaved water
15. 5 microliters buffer (3.1 for odd and cutsmart for even)
16. 1.5 microliters enzyme 1 (MluI for odd and EcoRI for even)
17. 1.5 microliters enzyme 2 (BspEI for odd and KpnI for even)
18. Vortex frozen DNA
19. Label microcentrifuge tubes for each digestion
20. Add 38 microliters mastermix to each PCR tube
21. add mastermix 1 to the odd numbered pS backbones and mastermix 2 to the even numbered pS backbones)
22. Add 12 microliters of the corresponding DNA
23. Spin down restriction digest solution in the tabletop microcentrifuge
24. Incubate the solution at 37 C for 30-60 minutes

PCR Amplify the Split Inserts

1. This process elongates the DNA using primers that Ben (our mentor) provided us to work with pS-14. We will purify the PCR later by running a gel in order to separate the DNA fragments that we want from the DNA backbone of pS-14 that we do not want.
2. Dilute forward and reverse primers from IDT according to IDT protocol.
3. Create working stock primers
4. Add 90 microliters deionized water to 8 microcentrifuge tubes labeled 1-8
5. Add 10 microliters primer from 100 micromolar stock to the appropriate tube
6. Label PCR tubes 1-8
7. Add 30 microliters deionized water to each PCR tube
8. Add 10 microliters 5x phusion HF reaction buffer to each PCR tube
9. Add 1 microliter dNTPs to each PCR tube
10. Add 2.5 microliters each of the primers (forward and reverse) (5 microliters of primer total)
11. Add approximately 15-20 nanograms DNA template
12. Concentration of pS14 was 431.4 nanograms/microliter so we diluted 1 microliter pS14 in 19 microliters of deionized water and then added 1 microliter of the diluted DNA to each tube
13. Add .5 microliter phusion enzymes
14. Put in thermocycler; setting: iGEM PCR:
15. Heated lid 105 C
16. Preheat lid off
17. Initial denaturation temp = 98 C for 1 minute (hot start off)
18. Number of cycles = 35
19. Denaturation = 98 C for 10 seconds
20. Annealing = 55 C for 20 seconds -temp depends on the primer
21. Extension = 72 C for 15 s
22. calculated according to bp length: ~1 kb= 15 sec
23. Final extension = 72 C for 10 minutes
24. Hold = 4 C forever

Gel Purification

Next, run the PCR products on a gel to separate the insert from the primer dimers

This process elongates the DNA using primers that Ben (our mentor) provided us to work with pS-14. We will purify the PCR later by running a gel in order to separate the DNA fragments that we want from the DNA backbone of pS-14 that we do not want.

1. Add 1g of Agarose Gel Mix to an Erlenmeyer flask
2. Add 100mL 1x TAE buffer to the flask
3. Microwave for about one minute until the gel mix starts to dissolve
4. Using heat protection, swirl the Erlenmeyer flask to cool the solution
5. Once the gel mix is dissolved completely and the solution is no longer steaming heavily, add 7.5 microliters Ethidium Bromide
6. Pour gel into the appropriate mold and let cool until it is slightly opaque
7. Place gel into gel box
8. Load 2 Log DNA Ladder into the right-most well of the gel
9. Add loading dye to the sample you wish to run on a gel
10. Load samples
11. Run the gel for 10-30 minutes at 135mV and 400A
12. Remove the gel from the gel box
13. Take a photo of the gel with the UV light imager
14. Cut band of gel using razor blade on UV light with face protection based on approximation of how long band digested fragment should be
15. Add about 600 microliters of GX per gel piece; 200 microliters for smaller pieces
16. Incubate in 70 C bead bath for 5-10 min until gels are melted
17. Follow your Gel Extraction Kit procedure (they vary by manufacturers)

Digesting Our Two Integrases with Enzymes in Order to Ligate

1. Add 30 microliters DNA to each tube labeled appropriately 1-8
2. Add 5 microliters buffer
3. Add 1.5 microliter of MluI for 1-4 and KpnI for 5-8
4. Add 1.5 microliter of BspEI for 1-4 and EcoRI for 5-8
5. Add 12 microliters autoclaved water
6. Vortex gently on the padded vortex
7. Incubate 20-30 min in order to get the enzymes to digest
8. PCR Purification of Our Restriction Results to Get the Integrase Inserts
9. Add solution from incubation to pellets in the mantel appropriately labeled
10. Add 500 microliters PX
11. Miniprep protocol beginning from wash buffers
12. Measure the concentration of DNA insert to EB on the nanodrop

Ligation

*Sulfuric smell means ligase buffer is still good

1. Plug into Ben Weinberg’s Ligation calculator which accounts for Mass Vec, Insert Size, Plasmid Size, Concetrations of Plasmid DNA, Concentrations of Insert DNA and returns the amount of Plasmid DNA, Insert DNA and water to add
2. Add 50 microliters into each tube of Top 10
3. Add 0.5 microliter T4 DNA ligase
4. Add 1 microliter 10x T4 ligase buffer
5. Add insert, backbone and water as specified by Ben’s spreadsheet
6. Vortex and spin down ligation products
7. Let the ligation mixture sit at room temperature for 10 minutes in order to let the backbones, inserts and solution ligate
8. add 50 microliters KCM to the Top 10 cells, the KCM should be 1x KCM
9. Add 10 microliters of ligation reaction mix
10. Place in thermocycler to cool for about 10 minutes then heat shock
11. Plate the solution, about 30 microliters of solution and spread per ½ plate
12. Incubate the agar plate in the 37 C overnight

Picking Colonies

1. Open flame
2. Fill deep wells will 2mL LB+carb per well
3. Choose three colonies as far apart from each other as possible
4. Pick the colony with the pipette tip and then drop the tip into the well
5. Leave the pipettes in each well until you are ready to place breathable film over the deep well
6. Throw pipette tips out into the sharps container
7. Grow colonies in deep well plates with LB + Carb overnight

Purifying the DNA from the Grown Up Colonies

1. Decant the media
2. Miniprep from the Deep wells

Test Cut

*Verifying that the insert went into the backbone* Use the same enzymes that we used to put the enzyme in, in order to cut the insert out so that the insert size will be the same on the gel*

1. 6.5 microliters of water
2. 1 microliter 10x buffer
3. 0.25 enzyme 1 (BSPE1 or KPN1)
4. 0.25 enzyme 2 (MLU1 or EcoRI)
5. *we made two master mixes of #plasmids +2 in order to have enough to pipette*
6. Vortex and spin down
7. Incubate at 37 C for about 30 minutes
8. Add loading dye, run on gel for 15 minutes