Difference between revisions of "Part:BBa K4441005"

 
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<partinfo>BBa_K4441005 short</partinfo>
 
<partinfo>BBa_K4441005 short</partinfo>
  
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129 150 22 60.59 -4.29 -5.35 41 ATCAGTTTGAACAGTTGTCTGG
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Sequence used in wetlab experiments: TCTGACTGAAAGCTGTATGGis taken from [1]
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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This is the sequence for the outer backward primer. The outer backward primer consists of a B3 region which is complementary to the B3c region of the template sequence [2]. The outer primer B3 hybridizes to B3c region of the target DNA and extends, displacing the BIP linked complementary strand and resulting in the formation of a dumbbell shaped DNA [2]. This B3 primer is part of the primer set that detects the BRAF V600E:
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TTACTTACACGCCAAGTCAATCATCCACAGAGACCTCAAGAGTAATAATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCTCGATGGA
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GTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGCACCAGAAGTCATCAGAATGCAAGATAAAAATCCATACAGCTTTCAGTCAGATGTATATGCATTTGGAATT
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GTTCTGTATGAATTGATGACTGGACAGTTACCTTATTCAAACATCAACAACAGGGACCAG
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====Dilutions====
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Storage Primer Concentration: 100 μM
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10X Concentration (Stock): 16 μM
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Volume of 10X primer mix: 10 μL
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1X Concentration (Final):1.6 μM
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Volume of storage primer needed: 0.2 μL
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Mixed with F3, FIP, BIP, LF and LB for a total of 4.4 μL -- Final primer mix
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1 μL of final primer mix is used in the LAMP reaction mix. For more information, visit https://2022.igem.org/Team:Washington
 +
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====Alternative Sequence====
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As this set of primer failed to produce a positive result, we used NEB LAMP Primer Design Tool (https://lamp.neb.com/#!/) to generate an alternative set of primers. Unfortunately, due to time and budgetary constraints, these primers have not been ordered and experimented with yet.
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Sequence: TCTGACTGAAAGCTGTATGG
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5′ pos: 200
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3′ pos: 21
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len: 20
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Tm: 56.40
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5′ dG: -4.76
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3′ dG: -4.23
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% GC: 45
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==Citations==
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1. Papadakis, G., Pantazis, A. K., Fikas, N., Chatziioannidou, S., Tsiakalou, V., Michaelidou, K., ... & Gizeli, E. (2022). Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples. Scientific reports, 12(1), 1-15.
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2. Loop mediated isothermal amplification - technote. (n.d.). Retrieved October 11, 2022, from http://www.premierbiosoft.com/tech_notes/Loop-Mediated-Isothermal-Amplification.html
  
 
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Revision as of 05:05, 12 October 2022


BRAF V600E B3

Sequence used in wetlab experiments: TCTGACTGAAAGCTGTATGGis taken from [1]

Usage and Biology

This is the sequence for the outer backward primer. The outer backward primer consists of a B3 region which is complementary to the B3c region of the template sequence [2]. The outer primer B3 hybridizes to B3c region of the target DNA and extends, displacing the BIP linked complementary strand and resulting in the formation of a dumbbell shaped DNA [2]. This B3 primer is part of the primer set that detects the BRAF V600E: TTACTTACACGCCAAGTCAATCATCCACAGAGACCTCAAGAGTAATAATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCTCGATGGA GTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGCACCAGAAGTCATCAGAATGCAAGATAAAAATCCATACAGCTTTCAGTCAGATGTATATGCATTTGGAATT GTTCTGTATGAATTGATGACTGGACAGTTACCTTATTCAAACATCAACAACAGGGACCAG

Dilutions

Storage Primer Concentration: 100 μM

10X Concentration (Stock): 16 μM

Volume of 10X primer mix: 10 μL

1X Concentration (Final):1.6 μM

Volume of storage primer needed: 0.2 μL

Mixed with F3, FIP, BIP, LF and LB for a total of 4.4 μL -- Final primer mix 1 μL of final primer mix is used in the LAMP reaction mix. For more information, visit https://2022.igem.org/Team:Washington

Alternative Sequence

As this set of primer failed to produce a positive result, we used NEB LAMP Primer Design Tool (https://lamp.neb.com/#!/) to generate an alternative set of primers. Unfortunately, due to time and budgetary constraints, these primers have not been ordered and experimented with yet.

Sequence: TCTGACTGAAAGCTGTATGG

5′ pos: 200

3′ pos: 21

len: 20

Tm: 56.40

5′ dG: -4.76

3′ dG: -4.23

% GC: 45

Citations

1. Papadakis, G., Pantazis, A. K., Fikas, N., Chatziioannidou, S., Tsiakalou, V., Michaelidou, K., ... & Gizeli, E. (2022). Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples. Scientific reports, 12(1), 1-15.

2. Loop mediated isothermal amplification - technote. (n.d.). Retrieved October 11, 2022, from http://www.premierbiosoft.com/tech_notes/Loop-Mediated-Isothermal-Amplification.html

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]