RNA was extracted from lab-safe E. coli cells used to test the LAMP reaction. Procedure was completed as described in Qiagen RNeasy Mini kit (1).

Genomic DNA extraction was performed during the preliminary stages of our project in order to test the LAMP protocol. Entire extraction was completed as described in Thermo Fisher GeneJET Genomic DNA Purification Kit (2).

• Thermo Fisher GeneJET Genomic DNA Purification Kit
• Bacterial RNA
• 50% Ethanol
• Centrifuge

1. Extract genomic DNA according to kit procedure.

PCR was carried out as proof-of-concept and to amplify our modified Bst gene fragment. PCR cleanup was completed as described in Thermo Fisher GeneJET PCR Purification kit.

• Stock dNTPs (10 mM)
• Template DNA
• Forward and Reverse Primers
• DNA Polymerase
• DNA Polymerase Buffer
• Betaine
• Thermal Cycler
• Thermo Fisher GeneJET PCR Purification Kit

1. Add reaction components in the following volumes:

• Forward Primer	0.5 μL
  Reverse Primer	0.5 μL
  DNA polymerase	0.5 μL
  DNA polymerase buffer	10 μL
  dNTPs	1 μL
  Template DNA	1 μL
  Betaine	13 μL
  Sterile Water	23.5 μL
  TOTAL	50 μL

2. Place reaction tube in thermal cycler for the following cycles:

• Cycle Step	Temperature (°C)	Time	Number of Cycles
  Initial Denaturation	98	1 min	1
  Denaturation	98	10 sec	30
  Annealing	primer dependent	30 sec	30
  Extension	72	DNA pol dependent	30
  Final Extension	72	5 min	1
  Termination	4	Indefinite	1

3. PCR cleanup reaction product according to kit procedure.

• Agarose Powder
• LB Buffer Solution
• Red Safe DNA Stain
• 10X DNA Loading Dye
• 10X DNA Ladder
• Gel Electrophoresis Chamber and Comb
• Microwave

1. Add 0.5 g of agarose, 50 mL of LB Buffer Solution, and 2.5 μL of Red Safe stain to an Erlenmeyer flask.
2. Heat the solution in microwave to 1 min and pour into gel electrophoresis chamber. Add comb to the chamber and allow gel to cool and solidify for 40 min.
3. Pipette 7 μL of DNA Ladder and DNA sample with 1 μL of DNA Loading Dye into separate wells.
4. Run gel at 120 V for 25 min to measure weight of DNA sample against DNA ladder.

• New England Biolabs WarmStart® LAMP Kit (DNA & RNA)
• LAMP Primers
• FIP
• BIP
• F3
• B3
• Loop F
• Loop B
• Purified Target RNA
• Hydroxy Naphthol Blue (HNB)

1. Prepare 10X LAMP primer mix with all primers in a single microfuge tube according to WarmSmart® LAMP Kit protocol.
2. Store primer mix at -20 °C for 30 min. Vortex primer mix for 5 sec.
3. Prepare 3 microfuge tubes for positive (all reagents), negative (no RNA template), and negative (no Master Mix). Thaw frozen reagents.
4. Follow modified WarmSmart® LAMP Kit Reaction Setup protocol.

• 	All Reagents	No RNA Template	No Master Mix
  LAMP 2X Master Mix	12.5 μM	12.5 μM	-
  Primer Mix	2.5 µM	2.5 µM	2.5 µM
  Target RNA	2 µM	-	2 µM
  HNB	0.5 µM	0.5 µM	0.5 µM
  dH2O	7.5 µM	9.5 µM	20 µM
  Total	25 µM	25 µM	25 µM

5. Vortex each sample tube and incubate at 65°C for 30 mins.

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