Experiments

Dilution standards:

Name	Concentration
Gene block	10 ng/ul
Primers	25 nmol/ul

SMMO Fragment PCR protocol:

1. https://www.neb.com/en-us/protocols/2012/08/30/protocol-for-q5-hot-start-high-fidelity-2x-master-mix-m0494
2. To a pcr tube add 2 ul of forward primer a and reverse primer a (10 uM), and 25 ul of Q5 Hot Start High-Fidelity 2X Master Mix.
3. Add 10 ul of 10 ng/ul template DNA fragment a, and 11 ul of milliQ or dH20 to the tube.
4. Repeat steps two and three in a second tube for fragment b and its associated primers.
5. Place both tubes in a thermocycler and run at the temperatures specified below.
6. To confirm successful amplification, take 10 ul from each tube into a separate tube and add 2 ul of Gel Loading Dye, Purple (6X), no SDS to each tube.
7. In the first well, place 10 ul of 1 kb Plus DNA Ladder for Safe Stains, in the subsequent to wells, place the 15 ul of prepared solution from the tubes with loading dye.
8. Run the gel and confirm that fragments a and b have visible lines at ~2100 and ~1100 bp respectively.
9. Confirm fragment concentrations with nanodrop.

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
30 Cycles	98°C 45-68°C 72°C	15-30 seconds 15-60 seconds 60(s) for fragment A, 30(s) for fragment B. - 30 second per kb
Final Extension	72°C	5 minutes

Plasmid fragment PCR protocol:

1. Repeat the Smmo fragment pcr protocol with new forward and reverse primers. You will need two sets of forward and reverse primers, one for the front end and one for the back end of the amye gene.
2. The fragments produced will be shorter, so the extension time in the pcr cycle can be reduced to one minute.

PCR Fragment purification:

1. https://zymoresearch.wufoo.com/forms/w1p5d1kg0a6kw05/
2. https://files.zymoresearch.com/protocols/_d4003t_d4003_d4004_d4013_d4014_dna_clean_concentrator_-5.pdf
3. Run all fragments that have been multiplied through pcr purification, measure their final concentrations with the nanodrop

DNA assembly protocol:

1. https://www.neb.com/en-us/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
2. Add a 10 ul of NEBuilder HiFi DNA Assembly Master Mix to a tube along with ~0.10 pico moles of each DNA fragment.
3. Add dH20 until the reaction volume is 20 ul.
4. Heat to 50 C for one hour.
5. Run a gel with 10 ul of the mixture to confirm the ligation of the fragments.

Competent cell prep (complex):

1. https://static.igem.org/mediawiki/2020/7/71/T--Brno_Czech_Republic--prot-test-com.pdf

Antibiotic IPTG plate prep:

Make IPTG stock (0.100mM - serves as 1000x for plates):

1. https://goldbio.com/product/1379/iptg
2. Weigh 0.238 g of IPTG
3. Add 10 ml sterile H2O. Dissolve completely.
4. Prewet a 0.22 µm syringe filter by drawing through 5-10 ml of sterile H2O and discard water.
5. Sterilize IPTG Stock through the prepared 0.22 µm syringe filter.

Make Spectinomycin stock:

1. Add 100 mg of spectinomycin to 1 ml of dh2O.
2. Vortex, then run to a 0.22 um filter to sterilize.

Make plates:

1. https://static.igem.org/mediawiki/2014/5/59/LMU_Munich14_Media.pdf
2. https://agscientific.com/blog/iptg-facts-protocols.html
3. https://www.addgene.org/protocols/pouring-lb-agar-plates/
4. Reheat 40 ml of premixed lb agar in a water bath and add 40 ul of IPTG stock (0.100mM) and 40 ul of Spectinomycin stock once the temp is ~ 60 C.
5. Stir to combine, then pour four plates.

Transformation protocol:

1. https://static.igem.wiki/teams/4417/wiki/protocols/bacillus-subtilis-transformation-protocol-created-by-ucl-igem-2022.pdf
2. Take a 500 ul sample of competent cells, and add 500 ng of ligated DNA to it in an sterile 1.5 ml tube.
3. Incubate at 37 C/250 rpm for 1 hour.
4. Spread mixture onto prepared spectinomycin plates, incubate at 37 C for 1-2 days.