Team:Stony Brook/Experiments

iGEM SBU 2021

Navigating to Experiments

Step	Directions
1	Start the annealing temperature at the higher Tm, go down 1 degree at a time for 1 cycle each until hit (lower Tm-5) and then back cycle that for 20 cycles. Start with 180s initial denaturation at 98 °C and end with 180s final extension at 72 °C.

Step	Directions
1	Weigh 0.5g agarose powder and 50ml 1X TAE buffer and add them to a flask.
2	Melt the mixture in a microwave until the solution becomes clear, and then add and mix EB or SYBR safe with the solution after the it cools to about 50~60 °C.
3	Pour the solution into the gel casting tray with appreciate comb.
4	Pull out the comb when the gel is completely solid.
5	Place the gel in the electrophoresis chamber and add enough TAE Buffer to submerge the gel.
6	Pipette DNA samples mixed with appreciate amount of loading buffer into wells on the gel.
7	Run the gel at 100V for about an hour.

Step	Directions
1	Set up the following reaction on ice, according to NEB:
2	Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
3	Transform NEB DH5-alpha or 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2 μl of the chilled assembled product, following the transformation protocol.

Step	Directions
1	Make a blank measurement by pipetting 1µl of water onto the NanoDrop and press blank on the NanoDrop program.
2	Wipe the droplet of water
3	Pipette 1 µl of DNA solution, close the NanoDrop and press measure.
4	Record and label the results.
5	Repeat for all DNA samples.

Step	Directions
1	Streak out the E. coli strain on an LB plate to isolate colonies and incubate at 37 °C overnight.
2	Use a sterile pipette tip to collect cells from a single colony and inoculate in 5 ml sterile liquid LB Medium at 37 °C overnight in a shaker incubator.
3	Pipette 1 mL cells to 100 mL liquid LB Medium at 37 ℃ shaker incubator until grow the cultures to OD600 reaching about 0.26.
4	Pipette 25 ml of the cells to a pre-chilled sterile 50 ml tube.
5	Use a centrifuge to rotate at 4 °C, 4000 rpm for 10 minutes.
6	Decant supernatant and resuspend the cells by 10 ml ice cold 100 mM MgCl2. Hold on ice for 30 minutes.

Step	Directions
1	Take competent cells out of -80°C and thaw on ice.
2	Warm up agar plates warm up to 37°C in the incubator.
3	Add of 2 μL vector into 50 μL competent cells, and GENTLY mix by flicking the bottom of the tube.
4	Incubate on ice for 20 min.
5	Heat shock each transformation tube in the 42°C water bath for 30 s.
6	Put the tubes back on ice for 2 min.
7	Add 950 μL SOC media or LB media(without antibiotic) to the bacteria and grow in a 37°C shaking incubator for 60 min.
8	Plate the transformation and incubate plates at 37°C overnight.

Step	Directions
1	The NEB protocol was followed.

Step	Directions
1	Protocol from Moore et al. paper was followed.