Difference between revisions of "Part:BBa K4479008"
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==F3 LAMP Primer for Oak Wilt V3==
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Loop-mediated Isothermal Amplification is a cheap and quick method to amplify a desired DNA target. LAMP uses four to six primers; F3, B3, FIP (Forward Inner Primer),  BIP (Backward Inner Primer), and the optional LF (Loop Forward) and LB (Loop Backward) primers. We are using the four primer system that recognises six regions on our target DNA and they will form dumbbell-shaped amplicons. Our lamp primers run at 71°C and they are deactivated at 95°C. The primers were designed with the GLAPD software, then ranked for competency using a computer model. This is the F3 Primer of our first set of LAMP primers designed for the MCM7 region of Bretziella fagacearum (MG270170.1).
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https://static.igem.wiki/teams/4479/wiki/parts-images/lamp-mcm7-g1-dna.jpg
 
https://static.igem.wiki/teams/4479/wiki/parts-images/lamp-mcm7-g1-dna.jpg
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==Characterization==
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===LAMP on qPCR===
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To show that the primer set is working and to verify the amount of time the assay requires to run, we conducted our LAMP test on a qPCR machine. A green fluorescent dye was added to the LAMP master mix for data collection. The total volume of each reaction is 25 uL. We also decided to run the assay with different concentrations of target DNA (10 uL, 5 uL, 1 uL, 0.5 uL, and 0.1 uL). The primers were mixed at a 7:7:2:2 ratio (F3:B3:FIP:BIP). The results showed that all the trials passed the threshold, meaning that amplification was a success.
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{| class="wikitable" style="float:right; margin-left: 10px; margin-right: 20px"
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|+ Time for Detection with Different Concentrations of Target DNA
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|-
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! Conc. (ng/uL) !! Time (min)
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|-
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! scope="row" | 10
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|24.65 ± 2.06
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|-
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! scope="row" | 5
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|25.64 ± 1.62
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|-
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! scope="row" | 1
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|25.02 ± 1.91
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|-
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! scope="row" | 0.5
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|29.60 ± 4.07
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|-
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! scope="row" | 0.1
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|35.17 ± 0.41
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|-
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! scope="row" | Average
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|27
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|}
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https://static.igem.wiki/teams/4479/wiki/wetlab-results/glapd1-amp.png
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To verify if the amplification was on target, we looked at the melting curve from the qPCR machine. There is only 1 peak and they are within the acceptable deviation range, so we conclude that the primer set is able to amplify the target gene successfully.
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{| class="wikitable" style="float:right; margin-left: 10px; margin-right: 20px"
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|+ Melting Temperature of Different Concentrations of Target DNA
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|-
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! Conc. (ng/uL) !! Temp (°C)
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|-
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! scope="row" | 10
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|88.97 ± 0.42
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|-
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! scope="row" | 5
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|89.05 ± 0.49
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|-
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! scope="row" | 1
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|88.63 ± 0.36
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|-
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! scope="row" | 0.5
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|89.11 ± 0.17
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|-
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! scope="row" | 0.1
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|89.15 ± 0.34
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|-
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! scope="row" | Average
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|88.98
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|}
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https://static.igem.wiki/teams/4479/wiki/wetlab-results/glapd1-melt.png

Latest revision as of 18:45, 13 October 2022

F3 LAMP Primer for Oak Wilt V3

Loop-mediated Isothermal Amplification is a cheap and quick method to amplify a desired DNA target. LAMP uses four to six primers; F3, B3, FIP (Forward Inner Primer), BIP (Backward Inner Primer), and the optional LF (Loop Forward) and LB (Loop Backward) primers. We are using the four primer system that recognises six regions on our target DNA and they will form dumbbell-shaped amplicons. Our lamp primers run at 71°C and they are deactivated at 95°C. The primers were designed with the GLAPD software, then ranked for competency using a computer model. This is the F3 Primer of our first set of LAMP primers designed for the MCM7 region of Bretziella fagacearum (MG270170.1).

lamp-mcm7-g1-dna.jpg

Characterization

LAMP on qPCR

To show that the primer set is working and to verify the amount of time the assay requires to run, we conducted our LAMP test on a qPCR machine. A green fluorescent dye was added to the LAMP master mix for data collection. The total volume of each reaction is 25 uL. We also decided to run the assay with different concentrations of target DNA (10 uL, 5 uL, 1 uL, 0.5 uL, and 0.1 uL). The primers were mixed at a 7:7:2:2 ratio (F3:B3:FIP:BIP). The results showed that all the trials passed the threshold, meaning that amplification was a success.

Time for Detection with Different Concentrations of Target DNA
Conc. (ng/uL) Time (min)
10 24.65 ± 2.06
5 25.64 ± 1.62
1 25.02 ± 1.91
0.5 29.60 ± 4.07
0.1 35.17 ± 0.41
Average 27

glapd1-amp.png

To verify if the amplification was on target, we looked at the melting curve from the qPCR machine. There is only 1 peak and they are within the acceptable deviation range, so we conclude that the primer set is able to amplify the target gene successfully.

Melting Temperature of Different Concentrations of Target DNA
Conc. (ng/uL) Temp (°C)
10 88.97 ± 0.42
5 89.05 ± 0.49
1 88.63 ± 0.36
0.5 89.11 ± 0.17
0.1 89.15 ± 0.34
Average 88.98

glapd1-melt.png