PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs). Candidate human protein DNAs were purchased from IDT as gBlock Gene Fragments. Bacterial expression plasmids encoding human protein fused to Cre were made using USER-cloning (New England BioLabs). Truncation of ProTα was done using blunt-end ligation to delete regions of ProTα. Following PCR, KLD enzyme mix (New England BioLabs) was used to phosphorylate and circularize the PCR product before transformation into NEB10beta cells (New England BioLabs).
Neb10beta cells
Manufactured by New England Biolabs
NEB10beta cells are a competent Escherichia coli (E. coli) cell strain developed for the efficient transformation of DNA. They are suitable for a variety of molecular biology applications, such as plasmid propagation and protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Kld enzyme mix, by New England Biolabs (1 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (1 mentions)
User cloning, by New England Biolabs (1 mentions)
Neon electroporation system, by Thermo Fisher Scientific (1 mentions)
Cd510b 1, by System Biosciences (1 mentions)
Quikchange, by Agilent Technologies (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
T4 dna ligase buffer, by Thermo Fisher Scientific (1 mentions)
Plasmid safe nuclease, by Illumina (1 mentions)
3 protocols using neb10beta cells
1
Cloning and Truncation of ProTα
2
Establishing CDK14 Knockout and Rescue Cell Lines
3
Golden Gate Assembly Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the Golden Gate reaction, initially 250 ng of each of the desired modules were mixed with 100 ng of the destination vector, 2 μl T4 DNA ligase buffer (ThermoFisher Scientific), 1.2 μl T4 DNA ligase (ThermoFisher Scientific) and 1 μl BSAI (New England Biolabs) and 0.2 μl BSA (New England Biolabs) in a total volume of 20 μl. In later experiments, the efficiency of the reaction was increased by combining equimolar amounts of modules (0.05 pmol) with 0.025 pmol of destination vector. The following program was then run on a thermocycler T100 or C1000 (BioRad): 50 cycles of 37 °C for 5 min and 16 °C for 5 min, followed by 37 °C for 30 min, 50 °C for 5 min, and 80 °C for 5 min. After the reaction was finished, 1 μl PlasmidSafe nuclease (Epicentre) and 0.85 μl of 25 mM ATP were added to each reaction, and the reactions were incubated at 37 °C for 60 min followed by 70 °C for 30 min, to remove any intermediate, not fully ligated products. 3 μl of each reaction were then transformed into chemically competent NEB10beta cells (New England Biolabs) and correct clones were selected on kanamycin-containing LB agar plates. Clones were analyzed via PmeI digestions and/or Sanger sequencing to confirm the presence of each individual expression cassette.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!