Team:NYU Abu Dhabi/Protocols

P R O T O C O L

Preparation of Agar Plates

  1. Measured 8.75g of LB Broth with Agar
  2. Measure 250 mL of MilliQ water in an erlenmeyer flask
  3. Poured LB Broth with agar into the flasks
  4. To autoclave, ensured the water is at the appropriate level
  5. Placed flask with foil covering opening and tape into the machine
  6. Turned on, close, lock, and choose mode 4 for agar
  7. Right before the autoclave was ready, took and thawed antibiotics
  8. Prepared the plates
  9. Once the autoclave was ready (near 50ºC), stopped the machine and carefully removed the flask with heat resistant gloves
  10. Placed 250µl of antibiotic in the flask and swirl
  11. Poured mixture into agar plates and allow to cool and solidify

Bacterial Transformation

  1. 4 tubes of NEB 5-alpha Competent E.coli cells were thawed on ice for 10 minutes.
  2. 4x5 µL of ligated DNA was added to a tube of E.coli cells. The tubes were carefully flicked 4-5 times to mix the cells and DNA.
  3. The mixtures were placed on ice for 30 minutes
  4. The mixtures were heat shocked at 42 °C for exactly 30 seconds.
  5. The mixtures were placed on ice for 5 minutes.
  6. 950 µL of SOC was pipetted into each mixture
  7. The mixtures were placed at 37 °C for 60 minutes. They were allowed to be shaken at 250 rpm.
  8. The cells were centrifuged and concentrated
  9. Each sample was spread across separate agar plates and left incubated overnight for 37˚C.

Inoculation

  1. Labeled 2 tubes Bd 2 and 2 with Bsal 4 (transformed product)
  2. Pipetted 6ml of LB broth into each tube
  3. Took respective bacteria colonies from agar plates and mixed with the broth
  4. Placed in shaker incubator (inoculated) for 18-24 hours at 37˚C.

Miniprep

Notes:
- Did not add optional LyseBlue reagant
- step (6) (7), and (8) were performed with centrifuge

  1. All centrifugation steps are carried out at 13000rpm in a table-top microcentrifuge
  2. Centrifuged bacterial overnight culture at **7830 rpm for 3** **minutes** at room temp.
  3. Liquid was removed so that only pellet was left in the tube.
  4. Pellet was resuspended in **250 µl Buffer P1** and was transferred into a microcentrifuge tube
  5. **250 µl of Buffer P2** was added and the tube was inverted **6 times** in order to mix the solution.
  6. Added **350µl of Buffer N3** and mixed immediately by inverting the tube 6 times.
  7. Centrifuged for 10 minutes at 13000 rpm.
  8. Applied 800µl of supernatant to the QIAprep **2.0 spin column**, **centrifuged** **for 30 seconds** and discarded the flow-through.
  9. Washed the QIAprep 2.0 spin column by adding **0.5 ml of Buffer PB, centrifuged for 30 seconds** and discarded the flow-through.
  10. Washed the QIAprep 2.0 spin column by adding **0.75 ml of Buffer PE, centrifuged for 30 seconds**, and discarded the flow through.
  11. Transferred the QIAprep spin column to the collection tube
  12. Centrifuged for 1 minute to remove residual wash buffer
  13. Placed the column in a clean 1.5 ml microcentrifuge tube.
  14. Eluted DNA with **50 µl of Buffer EB** to the center of the spin column and let it stand for 1 minute then centrifuged for 1 minute.
  15. The concentrations were measured using nano drop

Serial Dilutions

Calculations for Initial Dilutions (Bd and Bsal):

  • For Bd:
  • To go from 2*1010 to 1010 copies/µl
  • C1V1 = C2V2
  • V1 = (1010)(50µl)/(2*1010) = 25µl of Bd miniprep in 25µl do dH2O
  • For Bsal:
  • To go from 5*1010 to 1*1010
  • C1V1 = C2V2
  • V1 = (1010)(50µl)/(5*1010) = 10 µl of Bsal miniprep in 40µl dH2O

Serial Dilution Calculations

Copies/µl Measurements
10^10 See Specific Calculations Above
10^8 5µl of 10^10 in 495µl dH2O
10^6 5µl of 10^8 in 495µl dH2O
10^3 1µl 10^6 in 999µl dH2O
10^2 50µl of 10^3 in 450µl dH2O
10 50µl of 10^2in 450µl dH2O
1 50µl of 10^1in 450µl dH2O

PCR

  • Volume of template DNA used ~300ng
  • Annealing temperature set for Primer set 4 = 52 °C
  • Annealing temperature set for Primer set 1, 2 and 3 = 53 °C
  • Four PCR tubes (Bd2, Bsal4) were prepared in ice with the following reaction mixture:
    1. 25 µL Q5 High-Fidelity 2X Master Mix
    2. 1 µL 10 µM Fwd Primer
    3. 1 µL 10 µM Rev Primer
    4. 3 µL Template DNA
    5. 20 µL Nuclease-Free water
  1. The PCR tubes was gently mixed and provided a quick spin.
  2. The PCR tubes were transfered to a preheated thermocycler (95 °C)
  3. The following thermocycling conditions were set:
    • Initial denaturation: 95 °C for 30 seconds
    • 30 Cycles:
      • i. 95 °C for 30 seconds
      • ii. 52 °C for Primer set 4 and 53 °C for Primer set 2 for 30 seconds (2-5 degrees melting temp of primers)
      • 68 °C for 30 seconds
    • Final Extension: 68 °C for 5 minutes
    • Hold 4 °C
  4. The PCR tubes were taken when allowed to do so and stored at 20˚C.

Making an Agarose Gel

  1. Mixed 40 ml of 50X TAE buffer with 1960ml of MilliQ water to make 2L of 1X TAE buffer
  2. Measured out 2x 0.5g of Agarose
  3. Mesaured out 2x 50ml of 1X TAE buffer and poured into two conical flasks
  4. Mixed 0.5g of Agarose into each of the conical flasks with 1X TAE and gently swirled
  5. Microwaved mixtures in increments until boiling to dissolve the Agarose
  6. Poured solutions into two separate casting trays and placed the well combs into them, allowed them to set (around 1 hour)

Gel Electrophoresis

  1. Collected all the samples necessary (listed in table below) and placed them on ice
  2. Drop 1 µl of nucleic acid sample loading buffer for each sample labeled on parafilm (except on 100bp ladder)
  3. Drop 5 µl of each sample and mixed with loading buffer on parafilm.
  4. Placed set gel into electrophoresis chamber.
  5. Put samples into the wells of the chamber in order shown in table (Bd Gel/Bsal Gel)
  6. Poured 1X TAE buffer carefully into the chamber to cover the gel.
  7. Connected the electrophoresis chamber to MyRun electrophoresis.
  8. Ran gel on 135 V for 20 minutes.

CRISPR-Cas12a On PCR Product

  1. Pipetted 15µl of amplified DNA into a new labeled PCR tube.
  2. For 8 reactions (only 7 reactions were conducted, measurements for 8 reactions were used to compensate for any potential pipetting errors) the following were prepared in a PCR tube in this order:
    1. 16µL NEBuffer 2.1 (10x)
    2. 4µl 5µM gRNA (Bd or Bsal)
    3. 12µl 1µM cas12a (cpf1)
    4. Added 8µl FQ Quencher (50µM) into the CRISPR complex (this was only added when the 7 PCR tubes were ready for the reaction)
  3. Pipetted 5µl of the CRISPR complex into each of the PCR tubes containing the DNA/negative control
  4. Incubated the CRISPR-DNA complex at 37°C. The samples were checked every 5 minutes for 40 mins under UV

RPA

Reaction Mix for 1 RPA Lypholized Tube:

  • Primer forward (5uM) – 2.4 uL (1 RPA tube) → 4.8 uL (2RPA tubes)
  • Primer Reverse (5uM) - 2.4 uL (1 RPA tube) → 4.8 uL (2RPA tubes)
  • Rehydration Buffer – 29.5 uL (1 RPA tube) → 59 uL (2 RPA tubes)
  • Nuclease Free Water – 8.2 uL (1 RPA tube) → 16.4 uL (2 RPA tubes)
  • Mixed above components
  1. A 5uM dilution was made for each primer for RPA reactions (47.5 uL TE buffer, 2.5 uL primer)
  2. Added the reaction mix (42.5 uL) to 1 freeze dried RPA reaction tube
  3. 15 uL from above transferred to 2 separate PCR tubes (4 total from the 2 RPA reaction tubes)
  4. 1 uL of 280 mM magnesium acetate was added to each tube and mixed well to start the reaction
  5. 5 uL of DNA was added to each respective PCR tube
  6. 5 uL of nuclease-free water added to negative control sample
  7. Incubated for 20 minutes at 37 degrees Celsius
  8. Left at least 5µl for gel

CRISPR-Cas12a On RPA Product

  1. To prepare the CRISPR Complex, the following reagents in this order were added at room temperature to a single Eppendorf tube:
    1. 2uL (8uL for 4 reactions) NEBuffer 2.1 Reaction Buffer (10x)
    2. 0.5 uL (2uL for 4 reactions) 5uM gRNA
    3. 1.5 uL (6uL for 4 reactions) 1uM EnGen Lba Cas12a (cpf1)
  2. Incubated the CRISPR complex for 10 minutes at 37 degrees Celsius
  3. Added 1 uL (4uL for 4 reactions) of 50 uM FQ quencher to the CRISPR complex
  4. 5 uL of the CRISPR mixture was added to each RPA mixture (When the RPA mixture has finished amplifhying)
  5. Incubate for 20 minutes at 37 degrees Celsius
  6. Observe under UV Light every 5 minutes

RPA + CRISPR

  1. 5 uL of CRISPR complex prepared using the above method was added to four tubes with RPA mix (also prepared using the above method).
  2. Incubated for 20 minutes at 37 degrees Celsius.
  3. Observed under UV Light every 5 minutes.