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.
We gel purified both our backbone digestions and insert PCRs using agarose gel electrophoresis and an Epoch kit.
We PCR-purified our insert digestions using an Epoch kit.
We ligated together our compatibly-digested inserts and backbones to form newly cloned plasmids.
We transformed these ligation products into compatible E. coli K12 bacteria using heat shock. We plated our transformations onto LB agar plates overnight.
We picked colonies from our transformation plates and grew these up. We again used the Epoch Miniprep kit to purify these plasmids.
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
Add 18g LB Broth Mix (for 900 mL LB broth) to deionized water; measured with weighboats
Add 40g LB Agar Mix (for 1L LB Agar) to deionized water; measured w/weighboats
Mix contents of broth or agar until powder is dissolved
Placed in autoclave with autoclave tape on top, labeled each, loosened cap, run on liquid cycle at 120 C for 1 hour
Removed autoclaved solutions with heat gloves and set on counter to cool
Plating the agar
Start flame with bunsen burner
Layout plates flat on the table with the lids on
Use carbenicillin antibiotic mix 1000x; (added 600 microliter to each agar which was 600mL LB agar solution)
Mix agar solution
Using electronic pipette, add 20 mL agar to each plate, opening lid slightly, moving plate to cool
Switch pipettes after every 8 plates
Dispose of pipettes in their wrapper into sharps container
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
Open a flame
Use new pipette tip for each 50 mL tube
Label tubes
Resterilized opening and cap of stock LB broth before closing again
Growing Up Cultures
Inncoluate Plasmids from Frozen Stock
Have plasmids frozen at -80 C
Add 2 mL of LB media to a centrifuge tube
Prepare an ice tray or ice block for the frozen stocks to be held
Open flame
Scrape pipette tip on frozen cell stock and release the pipette tip into the appropriately labeled centrifuge tube of media
Immediately return frozen stocks to -80 C
Incubate plasmids in media overnight in 37 C 300rmp spinning incubator
Cell Stock Creation from frozen plasmids
All near an open flame
Label cryotubes for each bacterial cell stock
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)
Add 300 microliters of 10x glycerol to each cryo tube
Add 700 microliters of the bacteria cell stock into the appropriate cryo tubes
Store at -80 C
Miniprepping and Nanodropping
Miniprep
This process separates the plasmid DNA from the bacterial DNA so that we can use the plasmid DNA for our research.
Prepare the buffers according to the Miniprep Kit protocols that come with the Miniprep Kits.
Label the prepared buffers with the date that they were prepared and the initials of the researcher who prepared them.
Spin down the remaining bacterial cell stocks in the centrifuge tubes for 10 minutes at 3500 rpm.
Label microcentrifuge tubes while you wait: two microcentrifuge tubes, one intermediate and one final, for each bacterial cell stock
Prepare supernatant waste container by adding 10% bleach to an empty beaker
Pour supernatant from centrifuged tubes into supernatant waste container, so that only the pellet in the tube
Add 200 microliters MX1 buffer to each pellet
Resuspend the pellet by pipetting up and down
Add 250 microliters MX2 (this lyses the cells)
Flick bottom of tube to mix solution
Add 350 microliters MX3 within five minutes of adding MX2 (this neutralizes effects of MX2)
Flick bottom of tube to mix solution
Pipette about 900 microliters of pellet and solution into the labeled intermediate microcentrifuge tubes
Tabletop centrifuge all tubes at 1500 rpm for 10 minutes
Label Miniprep columns from Miniprep Kit
Insert Miniprep columns from Miniprep Kit into vacuum mantel
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
Add 500 microliters of WN into columns in columns
Let all of the WN pass through the mesh before adding WS
Add 700 microliters of WS into columns
Wait until all of the WS is gone before pulling out the columns
Turn off the vacuum
Insert the columns into their respective, labeled column holders
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)
Insert column into microcentrifuge tube labeled final
Pour excess WN and WS from the column holder into the supernatant waste container.
Dispose of the column holders into the biohazard waste
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
Spin down final microcentrifuge tubes with the columns in them at 1500 rpm for 1 minute
Dispose of the columns into the biohazard waste
Using the Nanodrop Fluorospectrometer
Wipe down surface of the laser and the detector
Clear settings with 1 microliter water (the result should yield NaN)
Blank the settings with 1 microliter EB (the results should yield 0)
Measure all DNA concentrations using 1 microliter of DNA diluted in EB
Digesting Backbones
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.
*keep the enzymes cold!*
Lookup optimal buffers for restriction enzymes on NEB
Digest the pS#odd backbones with MluI and BspEI and a 3.1 buffer
Digest the pS#even backbones with EcoRI and KpnI and a cutsmart buffer
Check which buffers are optimal
Check the timing and temperature required for digestion
Check the inactivation temperature
when using multiple enzymes in the same tube, use buffers that have optimal activity for both
Digest 3-4 micrograms DNA for a complete digest (there’s also a test digest, using less DNA)
The total volume of the solution should be 50 microliters
To make master mix multiply below ratios by 7 for 6 digestions (1 extra just in case)
*keep enzymes on ice to not risk inactivation
30 microliters deionized autoclaved water
5 microliters buffer (3.1 for odd and cutsmart for even)
1.5 microliters enzyme 1 (MluI for odd and EcoRI for even)
1.5 microliters enzyme 2 (BspEI for odd and KpnI for even)
Vortex frozen DNA
Label microcentrifuge tubes for each digestion
Add 38 microliters mastermix to each PCR tube
add mastermix 1 to the odd numbered pS backbones and mastermix 2 to the even numbered pS backbones)
Add 12 microliters of the corresponding DNA
Spin down restriction digest solution in the tabletop microcentrifuge
Incubate the solution at 37 C for 30-60 minutes
PCR Amplify the Split Inserts
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.
Dilute forward and reverse primers from IDT according to IDT protocol.
Create working stock primers
Add 90 microliters deionized water to 8 microcentrifuge tubes labeled 1-8
Add 10 microliters primer from 100 micromolar stock to the appropriate tube
Label PCR tubes 1-8
Add 30 microliters deionized water to each PCR tube
Add 10 microliters 5x phusion HF reaction buffer to each PCR tube
Add 1 microliter dNTPs to each PCR tube
Add 2.5 microliters each of the primers (forward and reverse) (5 microliters of primer total)
Add approximately 15-20 nanograms DNA template
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
Add .5 microliter phusion enzymes
Put in thermocycler; setting: iGEM PCR:
Heated lid 105 C
Preheat lid off
Initial denaturation temp = 98 C for 1 minute (hot start off)
Number of cycles = 35
Denaturation = 98 C for 10 seconds
Annealing = 55 C for 20 seconds -temp depends on the primer
Extension = 72 C for 15 s
calculated according to bp length: ~1 kb= 15 sec
Final extension = 72 C for 10 minutes
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.
Add 1g of Agarose Gel Mix to an Erlenmeyer flask
Add 100mL 1x TAE buffer to the flask
Microwave for about one minute until the gel mix starts to dissolve
Using heat protection, swirl the Erlenmeyer flask to cool the solution
Once the gel mix is dissolved completely and the solution is no longer steaming heavily, add 7.5 microliters Ethidium Bromide
Pour gel into the appropriate mold and let cool until it is slightly opaque
Place gel into gel box
Load 2 Log DNA Ladder into the right-most well of the gel
Add loading dye to the sample you wish to run on a gel
Load samples
Run the gel for 10-30 minutes at 135mV and 400A
Remove the gel from the gel box
Take a photo of the gel with the UV light imager
Cut band of gel using razor blade on UV light with face protection based on approximation of how long band digested fragment should be
Add about 600 microliters of GX per gel piece; 200 microliters for smaller pieces
Incubate in 70 C bead bath for 5-10 min until gels are melted
Follow your Gel Extraction Kit procedure (they vary by manufacturers)
Digesting Our Two Integrases with Enzymes in Order to Ligate
Add 30 microliters DNA to each tube labeled appropriately 1-8
Add 5 microliters buffer
Add 1.5 microliter of MluI for 1-4 and KpnI for 5-8
Add 1.5 microliter of BspEI for 1-4 and EcoRI for 5-8
Add 12 microliters autoclaved water
Vortex gently on the padded vortex
Incubate 20-30 min in order to get the enzymes to digest
PCR Purification of Our Restriction Results to Get the Integrase Inserts
Add solution from incubation to pellets in the mantel appropriately labeled
Add 500 microliters PX
Miniprep protocol beginning from wash buffers
Measure the concentration of DNA insert to EB on the nanodrop
Ligation
*Sulfuric smell means ligase buffer is still good
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
Add 50 microliters into each tube of Top 10
Add 0.5 microliter T4 DNA ligase
Add 1 microliter 10x T4 ligase buffer
Add insert, backbone and water as specified by Ben’s spreadsheet
Vortex and spin down ligation products
Let the ligation mixture sit at room temperature for 10 minutes in order to let the backbones, inserts and solution ligate
add 50 microliters KCM to the Top 10 cells, the KCM should be 1x KCM
Add 10 microliters of ligation reaction mix
Place in thermocycler to cool for about 10 minutes then heat shock
Plate the solution, about 30 microliters of solution and spread per ½ plate
Incubate the agar plate in the 37 C overnight
Picking Colonies
Open flame
Fill deep wells will 2mL LB+carb per well
Choose three colonies as far apart from each other as possible
Pick the colony with the pipette tip and then drop the tip into the well
Leave the pipettes in each well until you are ready to place breathable film over the deep well
Throw pipette tips out into the sharps container
Grow colonies in deep well plates with LB + Carb overnight
Purifying the DNA from the Grown Up Colonies
Decant the media
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*
6.5 microliters of water
1 microliter 10x buffer
0.25 enzyme 1 (BSPE1 or KPN1)
0.25 enzyme 2 (MLU1 or EcoRI)
*we made two master mixes of #plasmids +2 in order to have enough to pipette*
