Genome-wide CRISPR–Cas9 knockout (GeCKO) v2.0 library plasmids were kindly provided by Feng Zhang [14 (link)]. The GeCKOv2.0 library A consists of 65,383 single guide RNAs (sgRNAs) that target 19,050 genes and 1864 miRNAs, causing frameshift indel mutations that lead to loss-of-function alleles. MCF7 cells were transduced with lentivirus carrying the GeCKOv2.0 library at an MOI of 0.2–0.4 for 24 h to achieve 100 × coverage of each sgRNA construct and then selected with puromycin (3 μg/mL) for 7 days. Puromycin-resistant cells were expanded for another 10 days to allow gene editing. Transwell invasion assays were performed as described, and invasive and noninvasive cells were harvested. Genomic DNA was isolated from the cells using a Blood & Cell Culture Midi kit (Qiagen). The sequences targeted by sgRNAs were amplified by the two-step PCR method, and the primer sequences for lentiCRISPR sgRNAs are listed in Additional file 1 : Table S1 [14 (link), 15 (link)]. The PCR products were purified by agarose gel electrophoresis, quantified using a Qubit 3.0 Fluorometer (Invitrogen) and an ABI7900 real-time fluorescence quantitative PCR instrument (Applied Biosystems) and sequenced on a HiSeq 2500 instrument (Illumina) in single-end mode. The MAGeCK algorithm was used to analyze the FASTQ files and identify metastasis-related genes.
Blood cell culture midi kit
Manufactured by Qiagen
The Blood & Cell Culture Midi kit is a laboratory product designed for the isolation and purification of DNA from various sample types, including whole blood and cell culture samples. The kit utilizes a silica-based membrane technology to efficiently capture and purify DNA, making it suitable for a range of downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 instrument, by Illumina (1 mentions)
Slot blot system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Typhoon trio, by GE Healthcare (1 mentions)
Adaptor, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions)
Qiaprep plasmid midi kit, by Qiagen (1 mentions)
Hpaii, by Thermo Fisher Scientific (1 mentions)
5 protocols using blood cell culture midi kit
1
Genome-wide CRISPR Screening for Metastasis
2
DNA Methylation Analysis by Slot Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA from ESCs was extracted using the Blood & Cell Culture Midi Kit (Qiagen) according to the manufacturer's instruction. Anti-oxidant BHT (200 μM, Sigma) and deaminase inhibitor THU (200 μM, Sigma) were added to the lysis buffer and elution buffer. The Bio-Rad slot blot system was used according to the manufacturer's instruction. Nitrocellulose membranes (Amersham) were crosslinked, blocked with 5% milk and immunostaining was performed using a mouse monoclonal antibody against mC (Eurogentec, 33D3) or rabbit polyclonal antibodies against hmC, fC and caC (Active motif: 39791, 61233, 61224). Alexa488-coupled secondary antibodies were used for detection and the membranes were scanned with the Typhoon TRIO (GE Healthcare Life Sciences). Quantification was performed with ImageJ.
3
Genomic Sampling of Ant Castes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three colonies of A. echinatior (Ae322, Ae356 and Ae363) were collected in Gamboa, Panama, in 2006–2008, and maintained in the laboratory under standard conditions of ca. 25 °C and ca. 70% relative humidity where they were supplied with a diet of bramble leaves, rice and pieces of apple. RNA and DNA samples were collected from the three female castes: gynes, large workers and small workers. Animals were flash frozen in liquid nitrogen and the heads separated from the bodies with a pair of forceps. Heads from 50 gynes, 50 large workers or 200 small workers originating from the same colony were pooled and used for RNA extraction as described previously66 (link). The remaining body parts were likewise pooled per caste and colony, and subsequently used for DNA extraction (Supplementary Table 1 ). gDNA was extracted using a Blood & Cell Culture Midi kit (Qiagen) following the manufacturer’s instructions, with the modification that sample lysis was performed overnight.
4
Genome-Wide CRISPR Screen Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As previously described (20 (link)), genomic DNA was sequenced, and data were processed. First, 4 × 107 cells (i.e., >300× coverage over the GeCKO v2 library) were collected for genomic DNA extraction, using the Blood & Cell Culture Midi kit (13343; Qiagen). A total of 100 to 240 μg of genomic DNA (varying between replicates) were extracted, and sgRNA sequences were amplified by PCR using Herculase II Fusion DNA polymerase (600675; Agilent Technologies). Next, the amplicons were subjected to a second PCR amplification to attach Illumina adaptors and barcode samples. The primers used in the first PCR were as follows: F1, 5'-AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG-3’; R1, 5’-CTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCC-3’. The primers used in the second PCR were as follows: F2, 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-(variable-length sequence, up to 8 bp)-TCTTGTGGAAAGGACGAAACACCG-3’; R2, 5’-CAAGCAGAAGACGGCATACGAGAT-(8-bp barcode)-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTACTATTCTTTCCCCTGCACTGT-3’. DNA was sequenced on a HiSeq 2500 (Illumina) sequencer in rapid run mode, with a single-ended read length of 71 bp. After data acquisition, the number of reads that completely matched the GeCKO v2 library sequence was calculated.
5
Nuclear Extraction and Plasmid Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Note that 36 h after transfection, nuclei were extracted from the ESCs using the Blood & Cell Culture Midi Kit (Qiagen) according to the manufacturer's instructions. Plasmid DNA was re-isolated using the Qiaprep Plasmid Midi Kit (Qiagen). A total of 200 ng of re-isolated plasmid DNA was digested with 0.5 μl HpaII (Fermentas).
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!