Part:BBa_K4441009

BRAF V600E LB

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

Usage and Biology

This is the sequence for the Backward Loop Primer (LB). The LB primer is designed using the complementary strand corresponding to the region between B1 and B2 [2]. The Lb 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: ATCAGTTTGAACAGTTGTCTGG

5′ pos: 129

3′ pos: 15

len: 22

Tm: 60.59

5′ dG: -4.29

3′ dG: -5.35

% 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]