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Bio-protocol Preprint Repository
A preprint repository for life science protocols
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Request a detailed protocol from the "Materials and Methods" section of any research article.
Low-cost Medium-Throughput Protein Expression and Purification in E. coli
Q&A Published: Oct 29, 2026AbstractAs the cost of synthetic gene synthesis has decreased, more laboratories have access to large libraries of synthetic genes from which to express and purify proteins. However, current protocols for protein expression and purification are either too slow and laborious to work at large-scale, or require specialist equipment, such as robotic liquid handlers. Since many researchers work at a level between these extremes, we present a medium-throughput protocol to express and purify libraries ...
MicroC library preparation from Drosophila embryos
Q&A Published: Oct 27, 2026Abstract Micro-C maps chromatin contacts by digesting crosslinked chromatin with micrococcal nuclease (MNase) and ligating DNA ends that are held in proximity. This protocol describes library preparation from Drosophila melanogaster embryos, including embryo collection, sequential crosslinking, nuclei isolation, MNase digestion, end processing and biotin labeling, proximity ligation, removal of biotin from unligated ends, DNA purification, and sequencing library preparation. Biotinylated DNA is ...
Meiotic chromosome surface spreads
Q&A Published: Oct 23, 2026Preparation of Mouse Fetal Ovary spread for cytological analysis1. Collection of Embryonic OvariesC57BL/6J female mice were mated with male mice overnight. The following morning, females were examined for the presence of vaginal plugs. The day on which a vaginal plug was detected was designated embryonic day (E) 0.5. At E17.5, pregnant females were euthanized in accordance with the protocol approved by the relevant Ethics Committee. The uterus was removed, and the embryos were dissected and tran...
Construction of Pf-5 mutants and derivative strains
Q&A Published: Oct 21, 2026Protocol for making a deletion of an interested gene in the chromosome of strain Pf-5Day 1: Streak mutant Pf-5 strain on KB plate (no antibiotics) and grow overnight at 28°C.Streak S17-1 E. coli strain carrying deletion construct carried by pEX18Tc to be introduced on LB + Tc20 plate and grow overnight at 37°C.Day 2: Start 4 mL KB culture of Pf-5 strain and shake overnight at 28°C. Start a 4 mL LB + Tc10 culture of S17-1 strain and shake overnight at 37°C. (Note: The S17-1 strains tend to grow ...
In vivo model of liver metastasis by splenic injection.
Q&A Published: Oct 21, 2026Protocol for Establishing a Mouse Model of Colorectal Cancer Liver Metastasis by Intrasplenic Injection of MC38 Cells Followed by Splenectomy1. ObjectiveTo establish an experimental model of colorectal cancer liver metastasis (CRLM) by injecting MC38 murine colorectal cancer cells into the spleen of C57BL/6 mice, thereby introducing tumor cells into the liver through the portal circulation. Subsequent splenectomy is performed to minimize tumor growth within the spleen.This model is used to inves...
Single-molecule motility assays and TIRF microscopy imaging
Q&A Published: Oct 20, 2026Pre-made stock solutions 5XDLB with no KOAc150mM HEPES pH7.4 with NaOH10mM MgOAc 5mM EGTA50% glycerol 5X TEV50mM Tris.HCl pH8 750mM KCl50% glycerol 5X GF150 Buffer125mM HEPES pH7.4750mM KCl5mM MgCl2 300 nM Lis1 WT, del 298-308. Made MTs 488 LAST WEEK ProteinConc. Dim.Bufferdynein TMR from Aga K made on 01/27/2022- (A3-6) clear tubes197 nMGF150 buffer: 25mM HEPES pH7.4, 150mM KCl, 1mM MgCl2, 1mM DTT, and 10% glycerolDynactin unlabeledFrom 10/06/20 - Andrea's rack (B7-5)190 nMGF150 buffer: 2...
A. pullulans chemical transformation
Q&A Published: Oct 20, 2026A. pullulans Transformation Protocol (Published in DOI: 10.1083/jcb.202402114)NOTE: This protocol yields enough for 4-5 transformations. If you intend to do more, scale up and grow more cells. If you need less, extra comp cells can also be frozen at -80°C for several months and still work well with little to no loss in competency. Media and Solutions YPD Liquid for growing cellsFor 1L dissolve the following in 900 ml diH2O:10 g Yeast extract20 g peptoneSeparately, make 40% glucose Ad...
Isolation of embryos and stage 14 oocytes
Q&A Published: Oct 19, 2026Preparing Stocks for Collection Reagents:Media bottles kept at 4°CDry yeast Steps:Sprinkle dry yeast into the media bottles.Use CO2 to anesthetize flies, count flies.Stick the CO2 nozzle into the cotton of the bottle. Tip the bottle with the pipette tip on top of the bottle and tap on the bottom of the media bottle to cluster flies in one corner.Normally add 80 flies/ bottle for WT fliesCount 80 flies create pile. Use the pile as an approximation of # of flies to add to each bottle.After 2 days ...
Drug-tolerant persister generation
Q&A Published: Oct 19, 2026Generation of Drug-Tolerant Persister Cells by Prolonged Drug TreatmentBackgroundDrug-tolerant persister (DTP) cells represent a small fraction of cancer cells capable of surviving prolonged exposure to drug concentrations that strongly inhibit or eliminate most of the parental population. Drug tolerance can be transient and reversible, although surviving populations may also contain cells with more stable resistance mechanisms. This protocol describes a general approach for generating drug-tole...
Analysis of cell cycle and apoptosis
Q&A Published: Oct 15, 2026Protocol for the detection of apoptotic cells during cell cycle analysis. This method is described in the publication by Riccardi and Nicoletti, ‘Analysis of apoptosis by propidium iodide staining and flow cytometry’ (Nature Protocols, 2006).Principle of the method: apoptotic cells are characterized, amongst other things, by features such as fragmented DNA and, consequently, a reduction in nuclear DNA content. The fluorochrome propidium iodide (PI) binds (almost!) stoichiometrically to nucleic a...