EXPERIMENTS
OUR METHODOLOGY AND OTHER PROCEDURES FOLLOWED
OUR METHODOLOGY AND OTHER PROCEDURES FOLLOWED
Download the PDF version of our lab procedures, written by directors Vishwaa and Daniel, here:
| Day | Schedule |
|---|---|
| Wednesday | Transformation Blue White Screening |
| Thursday | Blue White Screening Select colony to grow in media (white colonies) |
| Friday | Mini-prep (19 tubes) Set up digestion for gel (if time) (maybe overnight) |
| Monday | Digest Run Gel Copies for Storage |
| Tuesday | PFAS Exposure |
| Wednesday-Friday | Time to redo parts, fix mistakes, etc. |
Bacteria Strain:
E. Coli DH5α™ Competent Cells
Protocol:
Thaw bacteria on ice ~ 20-30 mins
Gently mix cells
Aliquot 50 μl of e.coli in microcentrifuge tube *** label carefully
Add 1.5 μl of H20 into a different microcentrifuge tube
Pipette 0.5 μl of DNA (4ng/4ul) into H2O tube dilution
Mix.
Pipette the 2.0ul diluted DNA into the 50ul ecoli tube
Incubate the e-coli/DNA mixture on ice ~20-30 mins.
Heat shock each e-coli/DNA tube by placing bottom 50% - 66% of the tube in water bath 30 sec @ 42°C
Place e-coli/DNA tubes on ice ~2 min.
Add 250-1,000 μl Antibiotic-ABSENT LB or SOC media to bacteria tubes and grow in shaking incubator (document which media was used)
60 min @ 37°C 225 rpm
Pipette 10 and 20 μl e-coli/DNA mixture onto a 10 cm LB selective agar plate.
Gently spread bacteria throughout agar plate
Incubate plates @ 37°C O/N
Properly dispose and clean up
Note
3 instances in parallel for this protocol
Concentration of the DNA Plasmids 5 ng/ul
Both construct 1 and 2
Bacteria Strain:
E. Coli DH5α™ Competent Cells
Protocol:
Thaw bacteria on ice ~ 5 minutes
Aliquot 50 μl of e.coli in microcentrifuge tube *** label carefully
Add 1 μl of H20 into a different microcentrifuge tube
Pipette 0.5 μl DNA 1 (4mg/4ul) into H2O tube dilution + 0.5 ul DNA 2 (4mg/4ul)
Pipette the 2.0ul diluted DNA into the 50ul ecoli tube
Incubate the e-coli/DNA mixture on ice ~20-30 mins.
Heat shock each e-coli/DNA tube by placing bottom 50% - 66% of the tube in water bath
30 sec @ 42°C
Place e-coli/DNA tubes on ice ~2 min.
Add 250-1,000 μl Antibiotic-ABSENT LB or SOC media to bacteria tubes and grow in shaking incubator
60 min @ 37°C 225 rpm*** Document what media you end up using
Pipette 10 and 20 μl e-coli/DNA mixture onto a 10 cm LB SELECTIVE agar plate.
****Blue White Screening + selective agar in one plate
Gently spread bacteria throughout agar plate
Incubate plates @ 37°C O/N
Properly dispose and clean up
Bacteria Strain:
E. Coli DH5α™ Competent Cells / DNA +
Reagents:
X-Gal
Dimethyl sulfoxide (DMSO)
dH2O
Isopropyl β-D-1-thiogalactopyranoside, IPTG (100 mM)
Protocol:
Prepare 20 mg/ml of X-Gal in DMSO
5 ml of DMSO
100 μg of X-Gal
**Store at -20° C, wrap in foil
1 ml aliquots should be made of X-Gal in DMSO (completed up to here)
**Start plate preparation while bacteria is in the shaker**
Add ~6.5 µl of 20 mg/ml X-Gal solution per 1 ml of media on the surface of the selective agar plate
100 µl per plate
Add ~2.5 µl of 20 mg/ml X-Gal solution per 1 ml of media on the surface of the selective agar plate
40 µl per plate
Smear with spreader on top of the selective agar plate
Air dry plate in fume hood
Properly dispose and clean up
Bacteria Strain:
E. Coli DH5α™ Competent Cells / DNA +
Materials:
LB Broth
Antibiotics (Kanamycin, Ampicillin, Both)
15 ml Tubes
Toothpicks
Protocol:
Prepare 5 ml in each 15 ml tube
Make 5 tubes per transformation
Make 2 tubes per negative control
Pick a white colony for each tube using toothpicks and place it into a 15 ml tube
Keep at 37° C @225 RPM Overnight
Properly dispose and clean up
Bacteria Strain:
E. Coli DH5α™ Competent Cells / DNA +
Materials:
GeneJET mini-prep kit K0502
Protocol:
Add the provided RNase A solution to the Resuspension Solution and mix, can be used for 6 months when stored at 4°C
Add ethanol (96-100%) to the Wash Solution prior to first use, total volume of 55 ml
20 ml wash solution
35 ml Ethanol
If salt precipitation occurs on Lysis Solution and the Neutralization Solution
Redissolve any precipitate by warming the solution at 37°C
Cool to 25°C before use **Don’t shake too vigorously
Centrifuge selected colony (which was in media overnight) at 8000 rpm (6800 × g) in a microcentrifuge for 2 min at room temperature
Decant the supernatant and remove all remaining medium
Resuspend the pelleted cells in 250 µL of the Resuspension Solution
Transfer the cell suspension to a microcentrifuge tube
Bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain
Add 250 µL of the Lysis Solution
Mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear
Add 350 µL of the Neutralization Solution
Mix immediately and thoroughly by inverting the tube 4-6 times
Note: It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
Centrifuge for 5 min @>12000 × g, 10 000-14 000 rpm (table-top centrifuge should suffice) to pellet cell debris and chromosomal DNA
Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting Avoid disturbing or transferring the white precipitate
Centrifuge for 1 min @>12000 × g, 10 000-14 000 rpm (table-top centrifuge should suffice)
discard the flow-through
place the column back into the same collection tube
Add 500 µL of the Wash Solution to the GeneJET spin column
Centrifuge for 30-60 seconds @>12000 × g, 10 000-14 000 rpm (table-top centrifuge should suffice)
discard the flow-through
Place the column back into the same collection tube
**REPEAT (STEP 11)
Discard the flow-through
centrifuge for an additional 1 min @>12000 × g, 10 000-14 000 rpm (table-top centrifuge should suffice) to remove residual Wash Solution
**ESSENTIAL to remove ethanol
Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube
Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA
**Take care not to contact the membrane with the pipette tip
Incubate for 2 min at room temperature and centrifuge for 2 min @>12000 × g, 10 000-14 000 rpm
Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
Discard the column and store the purified plasmid DNA at -20°C
Properly dispose and clean up
Bacteria Strain:
E. Coli DH5α™ Competent Cells / DNA +
Protocol:
Add 400 μL of the overnight culture to 400 μL of 50% glycerol in a 1 mL screw-top tube/cryovial and gently mix
Make the 50% glycerol solution by diluting 100% glycerol in dH20
Snap top tubes are not recommended as they can open unexpectedly at -80°C
Freeze the glycerol stock tube at -80°C
Subsequent freeze and thaw cycles reduce shelf life
IMPORTANT: Visit this google sheet for complete exposure protocol and PFAS plate set up:
Google SheetsBacteria Strain:
E. Coli DH5α™ Competent Cells / DNA +
Reagents:
10uM PFOA Stock Solution
Protocol:
| Dilute PFOA Stock Solutions | Amount |
|---|---|
| 0 uM PFAS (Only liquid broth) | 5 ml |
| 20 ng/ml | 5 ml |
| 40 ng/ml | 5 ml |
| 60 ng/ml | 5 ml |
Collect all E.Coli/DNA+ cells in liquid broth 5ml
Centrifuge e.coli/DNA+ vials 5 min @ 1000rpm
Suction out all liquid broth
Isolating bacteria cells only
Pipette PFOA solutions into E.coli/DNA+ vial
Mix thoroughly by pipetting up and down
*** Try not to spill
Incubate vial 30min @ 37°C
Properly dispose and clean up