Mar 06, 2025

Public workspaceFUBP1/2/3 dTag protocol V.1

This protocol is a draft, published without a DOI.
  • Bercin Cenik1
  • 1Northwestern University
Bercin Cenik
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Protocol Citation: Bercin Cenik 2025. FUBP1/2/3 dTag protocol. protocols.io https://protocols.io/view/fubp1-2-3-dtag-protocol-d5am82c6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 06, 2025
Last Modified: March 06, 2025
Protocol Integer ID: 123949
Keywords: protocol for knock, protocol, terminal fkbp, loci with pitch system, resistance cassette, pitch system
Abstract
This is a protocol for knock-in of C-terminal FKBP-V with resistance cassettes into the FUBP1,2 and 3 loci with PITCh system.
Protocol materials
ReagentBbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L
ReagentrCutSmart BufferNew England BiolabsCatalog #B6004S
ReagentNew 6x Purple Loading DyeNew England BiolabsCatalog #B7024S
ReagentWizard SV Gel and PCR Clean-Up SystemPromegaCatalog #A9281
Troubleshooting
sgRNA cloning
3h
We will anneal following oligo duplexes into pX330-BbsI-PITCh:
FUBP1-dTag-sgRNA: BKC735_FUBP1_sgRNA1_F + BKC736_FUBP1_sgRNA1_R
FUBP2-dTag-sgRNA: BKC751_KHSRP_sgRNA1_F + BKC752_KHSRP_sgRNA1_R
FUBP3-dTag-sgRNA: BKC753_FUBP3_sgRNA1_F + BKC754_FUBP3_sgRNA1_R
Digest pX330-BbsI-PITCh:Duration02:00:00 Temperature37 °C thermal cycler
Amount3 µg pX330-BbsI-PITCh midiprep DNA
Amount3 µL ReagentBbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L
Amount3 µL ReagentrCutSmart BufferNew England BiolabsCatalog #B6004S
total to Amount30 µL with nuclease-free H20
2h
During digest, pour 0.7% TAE gelDuration00:30:00
30m
Briefly spin down PCR tubes to get rid of condensate.
Add ReagentNew 6x Purple Loading DyeNew England BiolabsCatalog #B7024S 1:6 to digested plasmid (ie 6ul for 30ul digest).

Run @150V for at least Duration00:30:00
30m
Gel purify digested fragment using ReagentWizard SV Gel and PCR Clean-Up SystemPromegaCatalog #A9281


Weigh gel slice and add binding buffer 1:1 w/v eg. 500ul buffer for 500mg of gel weight.

Dissolve gel slice Temperature55 °C , vortexing every 5 minutes to speed up dissolution. Continue until no chunks remain and slurry is completely liquid.