Difference between revisions of "Part:BBa K4441006"

Revision as of 05:12, 12 October 2022

BRAF V600E FIP

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

Usage and Biology

This is the sequence for the Forward Inner Primer. The FIP consists of a F2 region at the 3'end and a F1c region at the 5'end [2]. The F2 region is complementary to the F2c region of the template sequence. The F1c region is identical to the F1c region of the template sequence [2]. This FIP 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: 1.6 μL

Mixed with F3, B3, 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: TTCTCTGTAGCTAGACCAAAATCACTCCACAGAGACCTCAAGA

len: 43

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