Team:NYU Abu Dhabi/Protocols

Carried out on ice and by open flame, unless otherwise specified.

Add 3µl DNA to a microcentrifuge tube containing 50µl competent E. coli.
Ice for 20 minutes, followed by a 90 second heat shock in a water bath at 42˚C.
Return tubes to ice for 2 minutes.
Add 800µl SOC or LB broth.
Incubate on a shaker at 220 rpm and 37˚C for one hour.
Centrifuge for 1 minute at 13,000 rpm and discard 700µl of the supernatant.
Carefully resuspend the pellet and plate on agar containing the appropriate antibiotics. Spread gently using a glass spreader.
Incubate overnight (16 hours) at 37˚C.

This protocol was modified from the iGEM Ligation protocol
All materials are kept on ice unless otherwise stated.

Add the following reagents to a PCR tube:

2µL of digested plasmid backbone
equimolar amount of EcoRI-HF SpeI digested fragment (< 3µL)
equimolar amount of XbaI PstI digested fragment (< 3µl)
1µL T4 DNA ligase buffer
1µL T4 DNA ligase
ddH₂O until a total reaction volume of 20 µL

Incubate the ligation reaction at 4˚C overnight.
Transform with 3µL of product.

This protocol was modified from the iGEM Restriction Digest protocol
All materials are kept on ice unless otherwise stated.

Add the following reagents to a PCR tube:

150 ng of DNA to be digested (~3µL)
5µL of NEBuffer 2.1
1µL of the first enzyme (e.g. EcoRI-HF)
1µL of the second enzyme (e.g. PstI)
ddH₂O until a total reaction volume of 20µL

Mix well and spin down briefly.
Incubate the restriction digest at 37C for 1 hour, and then 65C for 10 min to heat kill the enzymes in a thermal cycler with a heated lid.
Run a portion of the digest on a gel (5µl) to check that both plasmid backbone and part length are accurate.

Insertion into pJET1.2 blunt cloning vector optimized protocol for Thermo Scientific CloneJET PCR Cloning Kit.

Run PCR on the gene of interest to approximately to obtain a DNA concentration of 500ng/µl.
The blunting reaction is setup on ice and composed of 10µl 2X Reaction Buffer, 500ng PCR product, 1µl DNA Blunting Enzyme and nuclease-free water up to 17µl. The reaction should be vortexed and centrifuged for 3-5 seconds and kept on ice.
Incubate the mixture at 70˚C for 5 minutes. Chill on ice.
The ligation reaction is setup on ice and composed of 1µl pJET1.2/blunt Cloning Vector and 1µl T4 DNA Ligase. This reaction should be added to the mixture from the previous steps. The reaction should be vortexed and centrifuged for 3-5 seconds.
Incubate the ligation mixture at room temperature for 30 minutes.
Transform the ligated mixture using the transformation protocol.

For any flask used, ensure that there is enough space to properly aerate the bacteria.

In a 15mL culture tube, add 5mL LB broth.
Using a sterile pipette tip, select a colony and drop it into the culture tube. Be careful not to let it hit the sides.
Loosely cap and incubate on a shaker at 220 rpm and 37˚C overnight (16 hours).

This is used for long-term bacterial storage and does not require re-transformation to obtain necessary cultures.

Add 700µl of the inoculated cultures to 300µl of 50% glycerol in a microcentrifuge tube.
Mix by inversion 4-6 times.
Store at -80˚C.
To plate, use a sterile pipette tip and scratch the surface of the stock and streak onto agar with appropriate antibiotics.

A more sensitive technique similar to Polymerase Chain Reaction. Control samples should be prepared carefully, as cross-contamination easily occurs due to the high sensitivity of LAMP. The following is the recipe for a 25µl reaction.

A set of 4 or 6 primers were designed using PrimerExplorer.
Make a stock containing Isothermal MasterMix and 20pmol inner primers and 5pmol outer primers.
Add 5µl DNA for amplification to 20µl stock.
PCR tubes should be placed in a thermocycler for 20 minutes at 65˚C.
The reaction can be stopped by placing the tubes on ice and visualized using 1X SYBR Green dye or any other appropriate dye.