Protocols

PCR

Set up PCR mixture as follow:
Set up thermocycler for PCR reaction as follow:
Add the other Component in the tube. If the added volume is less, pipetting up and down is necessary.
Mix reagents in tubes by pipetting the solution up and down slowly.
Quick spin the PCR tube to ensure the mixture is in the bottom of the tube.
Put it in the thermocycler (PCR machine) and set the cycle information.
Start the cycle and wait till it is finished.

Gel Electrophoresis

Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.
Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber.
Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times.
Load your samples and appropriate marker into your wells.
Incubate at 37°C and 250 rpm for 60-120 minutes.
Apply 130 volts to the chamber for 20 mins.
Check your gel using a transilluminator or other UV emitting device.
Re-suspend cells by light vortexing.
Plate resuspended cells as above.
Incubate overnight at 37°C with plates upside down.

DNA Gel Purification

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by trimming off extra agarose gel.
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (300 μl QG to 100 mg gel).
Incubate the tube at 60°C until the gel completely dissolved.
For DNA fragments <500 bp and >4 kb, add 1 gel volume of isopropanol to the sample and mix the reagents well.
o bind DNA, apply 800 μl sample to the QIAquick column, and centrifuge for 1 min.
Discard flow-through and place QIAquick column back in the same collection tube. It can be reused to reduce plastic waste.
Add 0.75 ml of Buffer PE to wash the QIAquick column, and centrifuge for 1 min.
Add 30 μl of distilled water to elute DNA.

Restriction Digestion

Prepare the reagents as follow:
Pipette the solution up and down to ensure all reagents are mixed well.
Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours.
Purify the DNA by PCR purification kit/gel extraction kit for downstream process.

Ligation

Prepare the reaction mixture as follow:
Allow the ligation to take place a 22-25°C for 10 minutes.
Send 5 μl of the ligated sample should be used for agarose gel electrophoresis to confirm whether ligation has occurred (Optional).

Preparation of Competent Cells

Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.
Spread the bacterial cell (DH5α) for plasmid replication on the LB.
Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm.
Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h).
Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes.
Collect the cell by centrifuging at 1000 g for 10 min at 4°C.
Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. * Cells must remain clod for the rest of the procedures!
Collect the cell by centrifuging at 1000 g for 10 min at 4 °C.
Centrifuge at 13,000 rpm for 1 min.
Gently resuspend the cell in each tube with 1.25 ml ice-old Ca/glycerol buffer.
Centrifuge at 13,000 rpm for 1 min.
Column dring with centrifuge 13.000rpm for 1 min.
Transfer all cells to one tube.
Dispense 100 μl aliquots of competent cells into each Eppendorf.
Store at -80 °C.
Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.

Transformation

Thaw 100µL competent E. coli cells on ice for 10 minutes.
Add:
- 10-20 µl DNA from a ligation reaction mix OR.
- 50-100ng DNA of a known plasmid.
Place the mixture on ice for 15 minutes.
Heat shock at exactly 42°C for 90 seconds.
Place on ice for 5 minutes.
Pipette 900 µl of room temperature SOC or LB media into the mixture.
Incubate at 37°C and 250 rpm for 60-120 minutes.
Pellet cells at 13000rpm for 1 minutes.
Remove and dispense 600 µl of supernatant.
Re-suspend cells by light vortexing.
Plate resuspended cells as above.
Incubate overnight at 37°C with plates upside down.

Miniprep

Harvest 3 mL cells at 1OD.
Centrifuge at 14,000 rpm for 30 s.
Discard the supernatant and resuspend the pellet with remnant.
Resuspend with 250 μl Resuspension Buffer.
Add 250 μl Lysis Buffer.
Add 350 μl Neutralization Buffer.
Centrifuge at 13,000 rpm for 10 mins.
Transfer supernatent to column.
Centrifuge at 13,000 rpm for 1 min.
Add 700 μl Washing Buffer B.
Centrifuge at 13,000 rpm for 1 min.
Column dring with centrifuge 13.000rpm for 1 min.
Inoculate with 30 μl distilled water.
Centrifuge at 13,000 rpm for 1 min.

Aptamer Folding Assay

Mix the following reagents
1. 30 ul 2x aptamer refolding buffer (20 mM Tris-HCl, 200 mM KCl, 10 mM MgCl2)
2. 1 uM aptamer RNA
3. 1 uM target RNA
4. 1.5 ul 200uM DFHBI
5. Nuclease free water up to 60 ul
Set up a control of aptamer only (30 ul buffer, 1 uM aptamer RNA, 1.5 ul 200 uM DFHBI, nuclease free water up to 60 ul)
Set up a blank (30 ul buffer, 1.5 ul 200 uM DFHBI, nuclease free water up to 60 ul)
Refold RNAs in 90 degree Celsius dry bath for 5 minutes
Incubate mixture in 37 degree Celsius for 45 minutes
Observe under blue light box and measure their fluorescence under CLARIOstar microplate reader at 447/501 Ex/Em.

Whole Cell Fluorescence Assay (T7)

Inoculate BL21 (DE3) transformed with the desired aptamer / probe to 5mL SOB.
Shake at 37C 250 rpm overnight.
Inoculate 1:50 into 10mL SOB with appropriate antibiotics.
Once OD600=0.4-0.6, add 1mM IPTG and shake for 2 hours.
Resuspend bacteria with M9 medium w/ 200uM DFHBI to final OD=0.2.
Incubate at 37C 250rpm for 45 min.
Measure fluorescence of the bacteria and blank (M9+DFHBI) with a plate reader.

In vitro Transcription

Perform PCR to attach a T7 promoter sequence (TAATACGACTCACTATAGG) to the target DNA.
Run agarose gel to confirm the size of the target DNA.
Perform gel purification of the desired band.
Measure DNA concentration using NanoDrop 2000.
Add 1ug of PCR product for a 20uL in vitro transcription using manufacturer’s protocol (NEB HiScribe™ T7 Quick High Yield RNA Synthesis Kit / T7 RiboMAX™ Express Large Scale RNA Production System).
Incubate at 37C for 4 hours..
Add DNaseI according to manufacturer’s instruction and incubate at 37C for 30 minutes.
Add 20uL of 3M sodium acetate pH 5.2 and top up with DEPC-treated water to 200uL.
Add 200uL of 1:1 phenol: chloroform (pH 8.0) and mix well.
Spin at maximum speed for 5 minutes and extract the upper aqueous layer.
Add 200uL of chloroform and mix well.
Spin at maximum speed for 5 minutes and extract the upper aqueous layer.
Add 400uL of absolute ethanol and mix well.
Incubate at -20C overnight or -80C for 1 hour.
Spin down at maximum speed at 4C for 30 minutes.
Decant the supernatant.
Rinse the pellet with ice cold 70% ethanol.
Use p10 pipette to aspirate all the supernatant carefully.
Air dry the pellet for 30 minutes to 1 hour in a RNA hood..
Resuspend the pellet with 50uL DEPC-treated water.
Measure concentration with NanoDrop 2000.
Make several aliquots and store at -80C.

Characterization: Specificity

Set 5 reactions for each 1 uM aptamer RNA with 5 different 1 uM target RNA. Example shown below is for PB2 aptamer, same can apply to N9, N2, H7 and H3 aptamer as well.
- 30 ul 2x aptamer folding buffer
- 1 uM PB2 aptamer
- 1 uM PB2 target/ 1 uM N9 target/ 1 uM N2 target/ 1 uM H7 target/ 1
- 1.5ul DFHBI
- Nuclease water top up to 60 ul
Set up a blank
- 30 ul 2x aptamer folding buffer
- 1.5ul DFHBI
- Nuclease water top up to 60 ul
Follow procedures of Aptamer Folding Assay

Detection Limit of RNA Aptamer

Set 6 reactions for each 2 uM aptamer RNA with 5 different concentration of target RNA.
- 30 ul 2x aptamer folding buffer
- 2 uM aptamer
- 1.5 uM/ 1 uM/ 0.5 uM/ 0.2 uM/ 0.1 uM/ 0.05 uM target
- 1.5ul DFHBI
- Nuclease water top up to 60 ul
Set up a control of aptamer only (30 ul buffer, 1 uM aptamer RNA, 1.5 ul 200 uM DFHBI, nuclease free water up to 60 ul)
Set up a blank (30 ul buffer, 1.5 ul 200 uM DFHBI, nuclease free water up to 60 ul)
Follow procedures of Aptamer Folding Assay

Characterization: Ions Dependency

Prepare the following 2x aptamer folding buffers of different ions concentrations
Follow procedures of Aptamer Folding Assay

Optimization of Aptamer Folding Condition

Mix the following reagents to 96 well plate
- 30 ul 2x aptamer folding buffer
- 1 uM aptamer RNA
- 1 uM target RNA
- 1.5ul DFHBI
- Nuclease water top up to 50 ul
Set up a control of aptamer only (30 ul buffer, 1 uM aptamer RNA, 1.5 ul 200 uM DFHBI, nuclease free water up to 60 ul)
Set up a blank (30 ul buffer, 1.5 ul 200 uM DFHBI, nuclease free water up to 60 ul)
Put the plate into real-time PCR machine
Set up thermocycler as follow:
- 95 degree Celsius for 5 minutes
- 94 degree Celsius for 30 seconds, decrement temperature by 1 degree Celsius per cycle, total 90 cycles
  + Plate read
- Melt Curve 4 degree Celsius to 95 degree Celsius, increment 0.5 degree Celsius every 5 seconds
  + Plate read
Start the cycle and wait till it is finished.

RNA-Snap Bacterial total RNA Extraction

Harvest 10⁸ E. coli cells at 14,000 xg for 30s.
Remove supernatant by aspiration.
Resuspend the pellet in 90uL of RNA extraction solution (10mM EDTA, 0.025% SDS, 1% 2-mercaptoethanol, 95% formamide) by vortexing.
Incubate the cell at 95 degrees Celsius for 10 minutes.
Spin down cell debris at 14,000xg for 5 minutes.
Transfer supernatant to a fresh tube carefully.
Add 360uL of RNase-free water and 50uL of NaOAc pH 5.2 to the supernatant.
Add 1.5mL of ice-cold ethanol to the RNA.
Incubate at -20C overnight or -80C for 1 hour.
Spin down at maximum speed at 4C for 30 minutes.
Decant the supernatant.
Rinse the pellet with ice cold 70% ethanol.
Use p10 pipette to aspirate all the supernatant carefully.
Air dry the pellet for 30 minutes to 1 hour in a RNA hood.
Resuspend the pellet with 20uL DEPC-treated water.
Measure concentration with NanoDrop 2000.
Run at least 2ug of total RNA in 1% agarose gel to confirm band intensity of rRNAs.
Store at -80C.

Team:Hong Kong-CUHK/Model

Protocols