Team:NYU Abu Dhabi/Results

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Results

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PCR

PCR reactions were run with designed primers to confirm the accuracy of the primers and also characterize the DNA fragments we would be working with. The agarose gel

Figure 1: Gel showing PCR amplification of gbpA, invA and lmo0733 gene fragments with designed primers. (Lane 1) 500 bp DNA ladder; (Lane 2) lmo0733 + lmo0733 primers; (Lane 3) nuclease-free water + lmo0733 primers; (Lane 4) gbpA with restriction sites + gbpA primers; (Lane 5) nuclease-free water + lmo0733; (Lane 6) gbpA + gbpA primers; (Lane 7) nucleas-free water + lmo0733 primers; (Lane 8) invA + invA primers; (Lane 9) nuclease-free water + invA primers

LAMP

Figure 2: Gel showing LAMP amplification of invA, gbpA and lmo0733 miniprep DNA with designed LAMP primers (PrimerExplorer). Amplification is seen for lmo0733 and gbpA but not invA when gene transformed E. Coli colonies were used. (Lane 1) 500 bp ladder; (Lane 2) invA miniprep + invA LAMP primers; (Lane 3) Nuclease-free water + invA LAMP primers; (Lane 4) invA transformed E. Coli colony + invA LAMP primers; (Lane 5) gbpA miniprep + gbpA LAMP primers; (Lane 6) Nuclease-free water + gbpA LAMP primers; (Lane 7) gbpA transformed E. Coli colony + gbpA LAMP primers; (Lane 8) lmo0733 miniprep + lmo0733 LAMP primers; (Lane 9) Nuclease-free water + lmo0733 LAMP primers; (Lane 10) lmo0733 transformed E. Coli colony + lmo0733 LAMP primers.

Figure 3: Gel showing LAMP amplification of gbpA with non colorimetric reaction mastermix (MM) (Optigene) with either hydroxy naphthol blue (HNB) or SYBR green added and with colorimetric reaction mastermix (NEB). (Lane 1) 500 bp ladder; (Lane 2) gbpA + Optigene MM + gbpA LAMP primers + HNB; (Lane 3) nuclease free water + Optigene MM + gbpA primers + HNB; (Lane 4) gbpA + Optigene MM + gbpA LAMP primers + SYBR green; (Lane 5) Nuclease free water + Optigene MM + gbpA LAMP primers + SYBR green; (Lane 6) gbpA + NEB MM + gbpA LAMP primers; (Lane 7) Nuclease free water + NEB MM + gbpA LAMP primers.

Sensitivity

RPA (50 uL reaction)

Figure 4: Gel showing RPA amplification of lmo0733, invA and gbpA miniprep DNA and transformed E. Coli colonies. The light bands seen in the negative control lanes are primer dimers and proteins from the RPA reaction. (Lane 1) 100 bp ladder;; (Lane 2) lmo0733 miniprep + lmo0733 RPA primers; (Lane 3) lmo0733 transformed E. Coli colony + lmo0733 RPA primers (25 uL reaction)

(10 uL reaction)

SYBR Green Visualization

LAMP

SYBR Green optimization

Here will go engineering stuff

Here will go proof of concept

Here will go cost analysis