...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...aliquots of it and storing those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells...by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at ...with the volatile reagents in the list above. Procedure Option #1 - Solution D Protocol Before starting...

...mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400 mL Note: If your lab has pre-mixed... to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might...might want to use as much as a liter of LB in a 2 L Erlenmeyer flask. Using a sterile pipette tip or toothpick...For long term storage of the bacteria, you can proceed with Creating a Glycerol Stock . You can now isolate...

...BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs Watch the...Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water...the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing and...

...Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should...the highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal ...inferred by the differences in colony size. Figure 2: Cas9 expression in monoclonal cell lines generated...for different cell types. Before starting this procedure, be sure that the cell pool has been sufficiently...

...Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc. *Pro-Tip* This selection method results... be subjected to multiple freeze-thaw cycles. Procedure Before beginning, determine the optimal dose of...antibiotic. This is the beginning of the selection process, which will begin the selection of a stable cell...

... recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the... day to ensure that growth times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 ... Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations...has been absorbed by the agar. This usually takes 1-2 minutes. *Critical* Be careful not to rip or shred...incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four...pellet. The total weight of each pellet should be ~1-2 g. *Pro-Tip* Make sure to weigh the empty tube beforehand...Libraries Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please ...

...cooling to room temperature (~45 minutes). Method 2. Place mixed oligos in a PCR tube. Place tube in a... a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes...vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and plate...to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer design)....be performed enzymatically later. Experimental Procedure Digest and purify vector: While waiting for your...

...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine serum albumin (BSA) Triton...grow to the desired density before labeling. Section 2: Fixing and permeabilizing cells Gently aspirate the...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-...

... plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown below ...individual colonies and effective selection. 150 µL of 2 mg/mL Carbenicillin plated over-agar Plate shows less...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve...resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration...

...s) Buffer BSA (if recommended by manufacturer) dH 2 O up to total volume *Pro-Tip* The amount of DNA that... 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) *Pro-Tip* The amount...from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg ...loading dye Electrophoresis buffer Pipet tips Procedure Select restriction enzymes to digest your plasmid...

...streak #2. Using a third sterile pipette tip, toothpick, or sterilized loop, drag through streak #2 and spread... (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...

... g sodium chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired size - we usually...indicated, the antibiotic powder can be dissolved in dH 2 O. *Note: Carbenicillin can be used in place of ampicillin...low a concentration for selection. Negative Result 2: Neither Strains Grows If neither strain grows, it's...video below to get a quick idea of how the whole process works. Equipment Autoclave Water bath Pipetman ...will grow). However, it is also more expensive. Procedure Measure 37g of pre-mixed LB-agar powder per L ...autoclave tape will darken during the autoclave process if your sample has spent at least 10 min at 121...

...right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to 10 µL P20 2 to 20 µL P100 10 to 100 µL P200 20 to ...the P2 this represents the third decimal place. Procedure How to Pipette Now that you know the different...

...-24 bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer...completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3...creates primer dimers and disrupts the amplification process. When designing, if unsure about what nucleotide...

... overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix. Note...learn how to create bacterial glycerol stocks. Procedure Follow the steps for Inoculating an Overnight ...

...10μL reaction for 5X buffer) 0.5-1μL T4 DNA Ligase H 2 O to a total of 10μL Note: If the DNA concentrations...vector from ligating to itself during the ligation process. If the sticky ends on either side of the vector...ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do...

...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...subcloning), including design and experimental procedures. Protocols...will save you time in future steps. Experimental Procedure Digest your DNA: Set up restriction digests for...

...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create... suited for analyzing the gene’s function. The process is shown graphically in the following cartoon, ...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR and purify the PCR product: Run PCR to...self-ligating recipient plasmid backbone. Transformation: Proceed with the transformation according to the manufacturer...