Synthesized oligonucleotides (Twist Bioscience) were dissolved in Buffer EB (Qiagen). 16 ng/uL single-stranded pooled oligos were amplified with NEBNext High-Fidelity 2X PCR Master Mix (NEB) with the following PCR protocol: Denaturation 98°C for 30s, 8 cycles of 98°C for 10s, 63°C for 10s, 72°C for 15s, final elongation 72°C for 3 min 40 μg pTF vector containing sgRNA-E + F scaffold with U6 promoter and Cas9-P2A-Zeocin with EFS-NS promoter is digested with Esp3l (Thermo Scientific) in FastDigest Buffer (10X, Thermo Scientific) and 1 mM DTT in 100 uL (Chen et al., 2013 (link)). The digested vector is also dephosphorylated in FastAP Thermosensitive Alkaline Phosphatase (1U/μL, Thermo Scientific) in FastDigest Buffer (10X) in 200 μL volume. Purified pooled oligos were cloned into digested pTF vector using 2X Gibson Assembly Master Mix with 10 times molar ratio of pooled oligos to digested vector and transformed to Endura Electro Competent Cells (Lucigen) and plated on LB + Amp plates (Figure S1D ). 565 E.coli colonies per guide ratio (>500× coverage) was used to process the 4 Maxi-Prep (Qiagen) reactions. After bacterial transformation, the plasmid library was sequenced to verify uniform sgRNA coverage and minimal bias (Figure S1E ).
Fastdigest buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
FastDigest buffer is a solution used in molecular biology applications to facilitate the digestion of DNA by restriction enzymes. It is designed to optimize the activity and specificity of these enzymes, enabling efficient and reliable DNA fragmentation. The buffer composition promotes the optimal conditions for the enzymatic reaction, ensuring consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (2 mentions)
Maxiprep, by Qiagen (2 mentions)
Eb buffer, by Qiagen (2 mentions)
Synthesized oligonucleotides, by Twist Bioscience (2 mentions)
Esp3l, by Thermo Fisher Scientific (2 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (2 mentions)
Nebnext high fidelity 2x pcr master mix, by New England Biolabs (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Spriselect beads, by Beckman Coulter (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Fastdigest ncoi, by Thermo Fisher Scientific (1 mentions)
Fastdigest dpni, by Thermo Fisher Scientific (1 mentions)
Phusion u dna polymerase, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
Gel imager, by Bio-Rad (1 mentions)
Sau3ai fd, by Thermo Fisher Scientific (1 mentions)
Quickchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Phusion pcr master mix, by Thermo Fisher Scientific (1 mentions)
23 protocols using fastdigest buffer
1
Pooled CRISPR Library Cloning Protocol
2
Pooled CRISPR Library Cloning Protocol
3
cDNA Linearization for Subsequent PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A cDNA oligo was annealed to the RT primer, generating a short double-stranded region suitable for BamHI restriction. This strategy also prevents BamHI activities on other endogenous BamHI restriction sites. Next, BamHI were applied, creating linear cDNAs with adapters at both 5′- and 3′-ends to prime subsequent PCR amplification. Next, oligo annealing mixture (43 μl water, 6 μl 10 × FastDigest Buffer (Fermentas), 5 μl 20 μM Cut_oligo (5′- GTTCAGGATCC
4
Feline PKD1 Exon 29 RFLP Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR restriction fragment length polymorphism (RFLP) assay was performed on all 377 blood samples at the Department of Veterinary Internal Medicine of Iwate University. Genomic DNA was
isolated from heparinized whole blood using a DNA purification kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). Exon 29 of the feline PKD1 was partially amplified
by PCR, producing a 559-bp amplicon, according to the Lyons et al. [20 (link)] protocol. DNA restriction was performed by digestion of 5
µl of amplification product in a total reaction volume of 10 µl that contained 0.5 µl of FastDigest MLyl (Fermentas, Waltham, MA, U.S.A.)
and 10×FastDigest buffer (Fermentas) incubated at 37°C for 30 min, followed by inactivation of enzyme at 80°C for 5 min. The RFLP pattern was obtained by running the complete digestion
reaction on a 2% agarose gel.
isolated from heparinized whole blood using a DNA purification kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). Exon 29 of the feline PKD1 was partially amplified
by PCR, producing a 559-bp amplicon, according to the Lyons et al. [20 (link)] protocol. DNA restriction was performed by digestion of 5
µl of amplification product in a total reaction volume of 10 µl that contained 0.5 µl of FastDigest MLyl (Fermentas, Waltham, MA, U.S.A.)
and 10×FastDigest buffer (Fermentas) incubated at 37°C for 30 min, followed by inactivation of enzyme at 80°C for 5 min. The RFLP pattern was obtained by running the complete digestion
reaction on a 2% agarose gel.
5
Purification and Digestion of Phi29 DNA
6
Validating CRISPR Genome Editing via RFLP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ssODN-mediated modification of the GFP target sequence was verified by Restriction Fragment Length Polymorphism (RFLP) of a newly created silent restriction site. The target sequences were PCR amplified from purified gDNA using KAPA HiFi HS ReadyMix, then amplicons were cleaved with FastDigest NcoI (Thermo Fischer Scientific, Cat. No. FD0573) restriction enzyme in FastDigest Buffer (Thermo Fischer Scientific, Cat. No. B64) and incubation for 30 min at 37 °C. Cleaved amplicons were then resolved by gel electrophoresis, acquired on an AE-9000N E-Graph (Atto) gel documentation system and quantified with the ImageJ software.
7
Metabolic Pathway Cassette Excision and Insertion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pMevT4 was generated by PstI digestion of pMevT followed by re-ligation of the backbone using T4 DNA ligase and standard molecular biology methods, excising the metabolic pathway cassette (atoB, ERG13, and tHMGR). pMevT-murI RBS variants were generated by uracil-excision cloning of murI into pMevT with a diversity of eight different murI RBS sequences added via the PCR primers for the cloning fragments (Supplementary Table 11 ). pMVA1 was similarly generated by uracil-excision cloning of PCR fragments, introducing a constitutive J23100 promoter with a PCR primer (Supplementary Table 11 ). PCRs were conducted by standard procedures with Phusion U DNA polymerase (Thermo). Uracil-excision cloning was performed by approx. equimolar mixing of the respective purified PCR products (Supplementary Table 11 ) in a 20 μL reaction, including 2 μL FastDigest buffer (Thermo), 0.75 μL USER enzyme (NEB), and 0.75 μL FastDigest DpnI (Thermo). The reaction incubated for 60 min at 37 °C followed by 20 min at 25 °C, and was subsequently transformed into chemically competent E. coli TOP10 cells.
8
Cas12m Protein Expression and CRISPR Cloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genes of selected Cas12m proteins were synthesized and cloned into pBAD expression vectors (Twist Biosciences) fused with N-terminal Twin-Strep-10×His-MBP purification tag encoding sequences (pTK200-203, pTK210-211, TK216-217). Cas12m expression vectors containing a minimal CRISPR region, consisting of two repeats and a single spacer between them, were cloned using Golden Gate strategy (pTK205-208, pTK213-214, pTK218-219). Specifically, the fragments of the minimal CRISPR region with the native adjacent sequences (up to 300 bp downstream the last repeat) were ordered as gBlocks (IDT). A plasmid encoding Cas12m and fragments of its respective CRISPR region was incubated with FD BpiI (Thermo Fisher Scientific) and T4 DNA ligase (Thermo Fisher Scientific) in 1× FastDigest buffer supplemented with 0.5 mM ATP (Thermo Fisher Scientific) for 30 cycles at 37°C/5 min and 16°C/5 min, followed by 65°C 10 min. The plasmids were propagated in E. coli DH5α and purified using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). Descriptions and links to protein and plasmid sequences can be found in Supplementary Table S3 and S4 .
9
Plasmid Cleavage by Restriction Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro cleavage experiments used commercial restriction enzymes (Thermo Fisher Scientific). The restriction enzyme NotI or XbaI (0.1 μL) was mixed with the compounds in a FastDigest buffer (Thermo Fisher Scientific). It was then incubated at room temperature for 10 min, and an ApaI-linearized px458 plasmid was added and reacted at 37 °C for 15 min. The samples were mixed with a DNA loading buffer. The cleavage products were electrophoresed on 1% TAE agarose gel and imaged using a Bio-Rad gel imager.
10
Pf-AR-1 Genome Library Construction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
gDNA was extracted as described and partially digested with Sau3AI FD (Thermo Fisher Scientific). Sau3AI FD was diluted 300-fold in 1× FastDigest buffer (Thermo Fisher Scientific) and was applied to the reaction mixture for a 3,000-fold enzyme dilution. Digested DNA was size-separated via agarose gel electrophoresis and fragments of ∼10 kbp were extracted and purified using Zymoclean Large Fragment DNA Recovery Kit, subcloned in BamHI-digested pRU1097, and electroporated in E. coli K12 DH10B MegaX. Plasmid DNA of ∼14,000 E. coli transformant colonies was extracted, representing more than 99.99% theoretical coverage of the PfAR-1 genome.
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!