May 31, 2023
ATG3 construct cloning
- 1Team Hurley
Protocol Citation: lmstrong 2023. ATG3 construct cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj85zngk5/v1
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: Working
We use this protocol and it's working
Created: May 31, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82663
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350 NIH
Abstract
ATG3 cloning
Ligation Independent Cloning
Ligation Independent Cloning
Amplify gene using PCR with Q5 polymerase. Design primers with overhangs compatible with 1GFP and 2BT vectors. For mutants, design overlapping primers in opposing direction as if using around the horn. Use these to perform 2-step PCR, verifying each step via gel.
Gel extract correct size band and measure concentration
Digest vector with SspI at 37 C for 1 hour. After 1 hour add CIAP to vector digest and incubate at 37 for another 30 minutes
Gel extract vector digest. PCR clean up insert digest. Measure concentrations of both.
Mix vector and insert at 1:4 molar ratio for ligation with 1 µL NEBuffer 2.1 and 1 µL 100 mM DTT to a final volume of 10 µL. Add 0.3 µL of NEB T4 DNA polymerase, and immediately incubate for 30 seconds at 50°C
Transform 4uL of reaction into DH5alpha cells. Plate everything. Sequence colonies to ensure correct mutation.