To produce CRY variants, modified Q5 DNA polymerase-based mutagenesis was used. Briefly, primers were designed using CRY-WT-CLIP as the template by NEBaseChanger (New England Biolabs). The desired DNA was amplified by PCR as above. PCR products were purified by a gel extraction kit and then 100 ng purified products were mixed with 1 μL each of T4 DNA ligase buffer (10x), T4 DNA ligase, T4 Polynucleotide Kinase and DpnI (New England Biolabs), to heal nicks in the PCR products. Nanopure water was added to make the total volume 10 μL. After overnight incubation at room temperature, all 10 μL were used for transformation following the same protocol as above.
T4 dna ligase buffer 10x
Manufactured by New England Biolabs
T4 DNA ligase buffer (10x) is a concentrated solution used to facilitate the enzymatic ligation of DNA fragments by T4 DNA ligase. The buffer provides the optimal ionic conditions and cofactors required for the ligase to efficiently join the ends of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (3 mentions)
Nebasechanger, by New England Biolabs (1 mentions)
Dpni, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Dna clean concentrator kit, by Zymo Research (1 mentions)
Gel purification kit, by Zymo Research (1 mentions)
Bsai hfv2, by New England Biolabs (1 mentions)
3 protocols using t4 dna ligase buffer 10x
1
Generating Engineered CRY Variants
2
Standardized Molecular Biology Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise specified, all molecular biology protocols were performed using NEB’s High-Fidelity Phusion Master Mix for PCR, Invitrogen’s Gel Purification Kit for gel extraction and Zymo’s DNA Clean & Concentrator kit for DNA purification (5 μg loading capacity). Qiagen’s QIAprep Spin Miniprep Kit was used to isolate plasmid DNA from cell culture. 3’-Azido-3’-Deoxythymidine (Azidothymidine, AZT) was purchased from Sigma-Aldrich. 5’-Triphosphate -3’-Azido-3’-Deoxythymidine (AZT-TP) was obtained through US Biological Life Sciences. All nucleotide oligomers and gBlocks were ordered from Integrated DNA Technologies. Ligations were performed using New England Biolabs’ T4 DNA Ligase Buffer (10x) and T4 DNA Ligase (400,000 units/mL).
3
UMI Incorporation and DNA Ligation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UMI incorporated fragments were mixed with phosphorylated BsaI_DNA_Linker (in a molar ratio of 1:40 UMI/linker), 5 μl T4 DNA ligase Buffer (10X) (NEB), 2 μl (800U) T4 DNA ligase (NEB cat: M0202S) and 2 μl of BsaI-HFv2 (NEB cat: R3733L) in a final volume of 50 μl. The mixture was incubated at 37°C for 2 h, then heat inactivated at 80°C for 20 min. Post ligation and heat inactivation, undesired linear fragments were removed by addition of 2 μl (20U) ExoV (NEB cat:M0345L) and 10 μl of 10 mM ATP, and incubation at 37°C for 30 min followed by heat inactivation at 70°C for 30 min.
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!