...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 ...optimal fixation method will vary depending on the sample type and the protein of interest. You may need ...expected and specific, include a positive control sample that you know expresses the protein, such as cells...the protein of interest, and a negative control sample such as cells that do not express the protein of...

...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...complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions. You may want to try higher...monoclonal lines from the early polyclonal populations. Sample Data Figure 1: Generation of monoclonal cell lines...

... 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 the...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...gel extraction of either the original sample or the amplified sample, followed by reamplification of the...

... plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown below are...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...absorbed. The spreading of cells can be done in the same way as the antibiotic, using either a bent micropipette...over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on LB-agar using different...

...temperature between 50-60°C for your PCR primers. Step 2: Linearize Vector In this example, the vector is cut...20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) 2.5 mM 2 DTT (100 mM) 5 mM 1 BSA (10 μg/μl) 0.25 μg/μl 1 T4...your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing... reaction is now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, ...have different leader sequences, but operate on the same principle (homologous to the first G on 3'-5' strand...

...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...centrifuge to separate different components in a liquid sample Using a Light Microscope Learn about the parts ...

...s) Buffer BSA (if recommended by manufacturer) dH 2 O up to total volume Pro-Tips The amount of DNA that... 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) The amount of restriction...from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg ...double digest (digesting with two enzymes at the same time), you will need to determine the best buffer...are digesting a large number of plasmids with the same enzyme(s) (for instance, in a diagnostic digest)...

... 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. *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...growth. See our sample data section below for positive and negative test results. Sample Data In all cases...Transfer the sterile water (in our case 220 mL) to the same bottle and swirl to form a medium/agar colloid. ...will darken during the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape...the autoclave and run on a setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 ...

...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 ...pipette tip is so that the same pipette can be used for measuring different samples without cross contamination...contamination as long as the tip is changed between samples. Tips can come loose in a bag, or can come preloaded...on the Pipette Although each pipette performs the same function, the numbers are read differently on each...correct amounts of liquid. If you are pipetting the same amount of liquid into different tubes or into wells...

... overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix. Notes...before you place the sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods...glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the morning...

...-24 bases 40-60% G/C content Start and end with 1-2 G/C pairs Melting temperature (Tm) of 50-60°C Primer...primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too long (> 30...

...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...by restriction sites that are also present in the same orientation on your target vector. If you are not...a cDNA from one plasmid to another. However, the same technique can be used to move promoters, selectable...useful to see if one set of enzymes will work in the same restriction enzyme buffer (see (Link opens in a .... If you select enzymes that can function in the same buffer, it will save you time in future steps. Experimental...

...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...about making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that...restriction enzymes that can both function in the same buffer, as this will save time later. In our example...