Part:BBa_K4441008

BRAF V600E LF

Sequence used in wetlab experiments: ACTGTGAGGTCTTCATGAAGAA is taken from [1]

Usage and Biology

This is the sequence for the Forward Loop Primer (LF). The LF consists of the F2 sequence (at its 3’ end) that is complementary to the F2c region, and the same sequence as F1c region at its 5’ end [2]. The LF 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: 0.4 μL

Mixed with F3, B3, FIP, BIP 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: ACTGTGAGGTCTTCATGAAGAA

5′ pos: 54

3′ pos: 75

len: 22

Tm: 60.99

5′ dG: -5.05

3′ dG: -3.87

% GC: 41

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. A guide to lamp primer designing - primerexplorer. (n.d.). Retrieved October 12, 2022, from http://primerexplorer.jp/e/v4_manual/pdf/PrimerExplorerV4_Manual_1.pdf

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]