Oct 21, 2018
Golden Gate Cloning LVL 2
- 1iGEM Team Marburg 2018
Protocol Citation: Daniel Marchal 2018. Golden Gate Cloning LVL 2. protocols.io https://dx.doi.org/10.17504/protocols.io.uview4e
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: October 21, 2018
Last Modified: October 21, 2018
Protocol Integer ID: 17034
Abstract
This cloning protocol refers to the Marburg Collection
Guidelines
This cloning protocol refers to the Marburg Collection
Materials
MATERIALS
T7 DNA Ligase - 100,000 unitsNew England BiolabsCatalog #M0318S
nuclease free water
Esp3INew England BiolabsCatalog #
R0734S
10X NEB T4 DNA ligase bufferNew England Biolabs
Before start
Before start, the resistance and ori parts have to be digested with BsaI and purified!
Predigestion
Predigestion
Before start, digest the Resistance and Ori plasmide with BsaI.
Purify the Fragments with PCR Cleanup.
Reaction Setup on ice:
Reaction Setup on ice:
1. Add 20 fmol of TU’s.
2. Add 0.5 µL BsmBI.
3. Add 0.5 µL T7-Ligase.
4. Add 1 µL T4-Ligase Buffer.
5. Fill with Nuclease-free water to 10 µL.
Thermocycling conditions
Thermocycling conditions
30 Cycles of 5min 37°C / 10min 16°C
30 min. 37°C.
10 min. 80°C.
Hold 20°C.