Sep 28, 2022
Molecular Cloning
- 1Shenzhen Polytechnic;
- 2ShenZhen Polytechnic
Protocol Citation: Lizhen Zhu, Ruixin Lin, Xiaoqi Ye, Ruiyan zeng, 18814306949 Minkun Huang, 624787660 2022. Molecular Cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw59o9vmk/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: October 08, 2020
Last Modified: September 28, 2022
Protocol Integer ID: 42914
Abstract
SZPT-CHINA Experimental methods of molecular cloning
Guidelines
advisers WeiJia Wu
LiJun Zhang（PhD)
Yongjun Tang(PhD)
Materials
MATERIALS
Q5 High-Fidelity 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0492L
Nuclease-free WaterNew England BiolabsCatalog #E6317
NEBNext Second Strand Synthesis Enzyme MixNew England BiolabsCatalog #E7425
Nuclease-free WaterNew England BiolabsCatalog #E7764
T4 DNA Ligase Buffer (10X)Thermo FisherCatalog #B69
2×PCR Master MixSolarbio
Vector linearization and target fragment preparation
Vector linearization and target fragment preparation
Q5 DNA polymerase PCR system(50 µL )
25 µL Q5 High-Fidelity 2X Master Mix
2 µL 10μL Forward Primer
2 µL 10 µM Reverse Primer
1 µL Template DNA
20 µL Nuclease-Free Water
Note
After mixing gently, centrifuge to collect all the liquid to the bottom.
PCR Program
Enzyme digestion Connection
Enzyme digestion Connection
Enzyme digestion Connection
Enzyme digestion and connection system(10 µL )
3 µL Vector
1 µL Insert
1 µL T4 DNA Ligase Buffer (10X)
0.5 µL NEB Golden Gate Enzyme Mix (BsaI-HFv2)
4.5 µL Nuclease-free H2O
Connection system
Transformation
Transformation
Transformation
Take 50 µL of competent cells in a sterile Eppendorf tube.
Add 2~5 µL of the plasmid to be transformed into each tube, gently mix the plasmid and competent cells with a pipette tip, and ice bath for 25 minutes.
Heat shock the ice-bath mixture in a 42℃ water bath for 45s, do not shake the centrifuge tube.
Take out the ice bath and cool for 2 minutes.
Add 800 µL of non-resistant LB medium preheated to 37°C to each tube, shake gently at 37°C, 150r/min for 1h.
Centrifugally concentrate to 100μL, spread it on an LB plate containing antibiotics, and incubate inverted at 37°C for 12-16h.
Make a negative control at the same time (use distilled water instead of plasmid).
Colony PCR screening of positive clones
Colony PCR screening of positive clones
Colony PCR screening of positive clones
Add 25ul of sterile water to the 96-well plate, pick about 15-20 colonies from the successfully transformed DH5α bacterial plate with a pipette tip, and mix them evenly. (Make a corresponding mark on the picking plate).
Put the mixed bacteria solution into 10ul to 200ul PCR tube and mark it.
Take 0.1ul of bacterial solution in a 96-well plate and add it to the antibiotic-containing plate with a pipette tip and mark it.
After mixing, the bacteria solution is used for PCR, the system is as follows(20 µL )
5 µL 2×PCR Mix
0.5 µL 10μM Forward Primer
0.5 µL 10 µM Reverse Primer
2 µL Bacteria solution
2 µL dd H2O
The procedure is as follows
Verification result of 1% agarose gel electrophoresis.