This page contains common protocols used in the lab. 🔬microscope and cell culture booking ðŸ§«

⚖️  Analyze oligo:

🧬     Tm 🔥 :           length 📏 : g/mol       ÂµM

⚖️  Molarity Calculator
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* always use the batch-specific molecular weight of the product found on the vial label.

⚖️  Dilution Calculator
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Post-phosphoramidite conjugation to oligonucleotides on non-solid support

🧬  NHS ester labeling of amino moeities
🧬  Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) of azides to alkyne moeities
🧬  Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) of ADIBO to azide moeities

Protein labeling

🧬Antibody conjugation

Oligonucleotide purification

🧪 Silica spin column
🧪 PAGE purification
🧪 Ethanol precipitation
🧪 Enzymatic oligo ligation and cleanup

Synthesis

🧬 Fluorescently labeled RNA
🧬 siRNA synthesis by in vitro transcription

Silanization

🪟🔬 Amino functionalized coverslips

Extraction

🧫 Total RNA extraction in Paramecium

Paramecium

Nuclear and membrane co-staining of Paramecium

In situ library preparation

🧫  Targeted: Padlock probes on cDNA
🧫  Targeted: Padlock probes on mRNA
🧫  Untargeted: cDNA circularization.
🧫  smFISH
🧫  In situ proximity ligation assay (PLA)
🧫 DSS (disuccinimidyl suberate) crosslinking in situ

In situ sequencing

🔬 Sequencing-by-synthesis (SBS) using reversible dye-terminator
🔬 Sequencing-by-ligation (SBL) using DNA ligase and 5'phosphate ligation.

Live imaging

Transfection for live imaging
Bacteria transfection

Computational pipelines

🤖  Making presentations using Quarto, Markdown and RevealJS
🤖  RNA-seq alignment using bowtie
🤖  Read alignment and visualization using JBrowse2
🤖  Read annotation and feature counting using featureCounts
🤖  RNA-seq alignment using STAR
🤖  In situ transcriptome accessibility ex situ reads pipeline
🤖  In situ transcriptome accessibility in situ reads pipeline

Common lab buffers

🍶 0.5 M carbonate/bicarbonate (pH 8.75)
🍶 10x 1M Sodium acetate (pH 6.0)
🍶 10% Triton X-100
🍶 Formamide strip buffer
🍶 Dryl's buffer
🌾 Paramecium wheat grass medium
🍶 Washbuffer for in situ hybridization
🍶 DEPC-PBST