...the amplified sample, followed by reamplification of the DNA, but please note that NGS should be performed... Protocols CRISPR Library Amplification CRISPR Library Amplification You may also like... Pooled libraries...different genes in an organism's genome, for example. Amplification is usually necessary to produce sufficient... pre-amplified stock. A last option is to perform a gel extraction of either the original sample or the...Follow this protocol to perform amplification of CRISPR pooled plasmid libraries in Escherichia coli ...refer to our pooled library material pages for amplification protocols that have been developed by the depositor...available. If a pooled library does not yet have an amplification protocol, the following protocol can be used...

...empty NTC C AAV reference Sample 3 D E Sample 1 Sample 4 F E Sample 2 Sample 5 F Perform data analysis...most samples. If sample is expected to have a titer <1 x 10 12 GC/mL, use dilutions 3–6 If sample is expected...between them. Sample Data Figure 1: Example of a valid 8-point standard curve. Figure 2: Example of the amplification...standards and samples in duplicate at least If possible, include an AAV reference sample of known titer...the expected titer of the samples being tested. Always include a No Template Control (NTC), i.e master...Count the number of samples (n) and prepare master mix for an additional 10 samples (n+10 – the additional...that there is enough master mix for all samples). Each sample requires 15 μL of master mix. Pro-Tips Use...

...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can be used...constant across samples. In the RRE example below, 2-fold serial dilutions of a sample were loaded in ...tube as shown below. For 8 samples prepare enough master mix for 9 samples. Component Volume 9X Volume...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...ddPCR Lentivirus sample data, RPP30 positive (green) and negative (black) droplets Sample ID Plasmid ID ... higher dilutions depending on their particular sample. This protocol was modified from the publication...Starting Thaw the master mix, primers/probe mixes and samples on ice before use. Wipe down all pipettes and surfaces...

...of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample briefly...concentration of your samples based on your sample’s HC + LC area. Example calculation: If the sample HC + LC Area...250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample is separated by denaturing...Invitrogen NP0001 4X NuPage sample buffer, Invitrogen NP0007 10X NuPage sample reducing agent (500 mM dithiothreitol...microcentrifuge. Heat the samples for 10 min at 100 °C in a heat block. Spin the sample briefly in the microcentrifuge...one lane before loading samples. Load 20 µL of each recombinant antibody sample to the appropriate well... mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed...

...of DNA (the template), a researcher can create thousands of identical copies using a simple set of reagents...If you are using the same template DNA for all your reactions, the Template DNA can be added to the master...particularly for GC-rich templates. What does each ingredient specifically do? Template DNA: Contains the portion...interested in amplifying for analysis and manipulation. It is essential that the template DNA and primer...Hybridize and are complementary to the 3’ ends of the sense and anti-sense strands of the template DNA. They ...polymerases from other species. In addition to the template DNA and the Taq polymerase, PCR requires free ... and downstream (3’) of the DNA segment to be amplified. When these reagents are combined in an appropriate...

... 10 seconds. Incubate sample(s) for 15 minutes on ice and centrifuge the sample(s) for 15 minutes at 12,000...Always wear gloves, and whenever possible, keep RNA sample and reagents cold and work quickly to reduce RNA...use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for 5 minutes to ...to allow for dissociation of the nucleoprotein complexes. The effectiveness of your RNA isolation will ...how effective your cell lysis protocol is. While simple homogenization is effective for most mammalian ... tissues such as bone, or bacteria/yeast/plant samples will require additional steps to effectively lyse...open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. ...

...helps concentrate the light onto the sample, and then through the sample. However, some of that light won’...hitting the sample or b) using dyes or stains that add color to some components of the sample but not others... power objective to find your sample. Place your slide (or other sample type) on the microscope stage....so that the light shines onto your sample. To ensure that your sample is adequately illuminated, look through... coarse focus knob to bring your sample into focus. Once your sample is in focus, use the stage arm to... you can easily see your sample. Then use the focus knobs to bring the sample into focus. Pro-Tip As you...magnification used, any stains used in your sample, and sample information so that you or others can interpret...

... which you need to spin your samples. For example, spinning down samples in 1.5 mL microfuge tubes at ...liquid sample. It achieves this by using centrifugal force, which is generated by spinning the sample at ... with water to the same volume as your samples. If your samples are significantly higher density than ...centrifuge to separate different components in a liquid sample. Protocols...RNA extractions. Plus, centrifuges are useful for simply collecting liquid to the bottom of a tube. You ... containers, spin at different speeds, or keep samples at specific temperatures. This protocol will cover...for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background Information ...

...finite binding capacity. If your sample exceeds the capacity, divide the sample among multiple columns. For...4 mL of recombinant antibody sample. Pro-Tip If the volume of the sample is greater than 4 mL , then divide... Slowly apply the sample to the center of the compact resin bed. Pro-Tip For sample volumes <1.5 mL top...the sample with 0.2 mL PBS to aid protein recovery. Allow the resin bed to fully absorb the sample. Centrifuge...Spectrophotometer to see if the sample has reached the desired concentration. If the sample concentration is still... to see if the sample has reached the desired concentration. Gently transfer the sample from the reservoir... 250 mL sterile bottle, VWR 430281 0.45 µm PES complete filtration unit, 500 mL, rapid-flow, VWR 73520...

... ddPCR sample data Sample Dilution Concentration Titer (GC/mL) Average Titer (GC/mL) AAV Sample 1 6400000...tube as shown below. For 8 samples prepare enough master mix for 9 samples. ITR Master Mix Volume 9X Volume...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...prepared for each AAV sample, and the three final dilutions are assayed. The samples that are assayed are...across the dilutions. In the example below, 2-fold serial dilutions of a sample were loaded in wells A04,... higher dilutions depending on their particular sample. For additional tips on AAV titering using ddPCR... on the insert. Please ensure that you are in compliance with your institution’s biosafety regulations...

...correspond to sequences on the template molecule (must be complementary to template strand). However, primers...One needs to design primers that are complementary to the template region of DNA. They are synthesized ...primers do not need to correspond to the template strand completely; it is essential, however, that the 3’...3’ end of the primer corresponds completely to the template DNA strand so elongation can proceed. Usually...well. Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a... a long primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too...primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower...

...duplicate samples on the plate. Subtract the average absorbance of the blank from all of the samples. Plot...concentration of the sample from the standard curve. Determine the volume of sample required to load an...protein for each sample. Pro-Tip The ideal total protein loaded will vary between samples and target proteins...abundance in the sample you will need to load a greater amount of total protein. Prepare sample for loading...Dilute 4X protein loading buffer in the sample to 1X . Boil the samples for 10 min at 100 °C . Section 3: SDS-PAGE...blotting to visualize a protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel... the host species of the primary antibody. For example, use an anti-mouse secondary antibody for primary...

...to the stop codon (TGA, in this example). Assuming you are amplifying from plasmid DNA (rather than from..., but we need to use the reverse complement to get PCR amplification. We can start similarly, taking ...generate the reverse-complement of this sequence so that we can successfully amplify the ORF. You can generate... to be amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying from a... a plasmid or simple template, there is very little chance for mis-priming, so you can use a pretty wide...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...choice with minimal limitations. Background In its simplest form, PCR based cloning is about making a copy...

...steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, .... Loading Samples and Running an Agarose Gel: Add loading buffer to each of your DNA samples. Note: Loading...Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or...bands needed to be separated - see FAQs below . Simply adjust the mass of agarose in a given volume to...). Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, ...room temperature for 20-30 mins, until it has completely solidified. Pro-Tip If you are in a hurry, the...glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well...

...sterile dH20 to 40 μl Step 4: Amplify Insert by PCR Perform PCR amplification of your insert following the...through the transformation/replication process. LIC employs long overhangs to form a stable association between...replication cycle. Empty vectors for LIC typically employ type II restriction enzymes (e.g. BsaI), which ...counter-selection for negative clones as in the example shown here. The manufacturer of an LIC-specific...respective primers. Below we use pNIC28-Bsa4 as an example of LIC experimental design. Search Addgene's collection... Your Primers Primer design for LIC is often as simple as using the backbone manufacturer's suggested ...sequence is used, plus a minimum of 18 bp of your template sequence. 5' and 3' primers will have different...

... 50% amplitude. Return to ice between each round of sonication to avoid overheating of the sample. Mix... mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times...-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return...CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile ...sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous...0.08 μg/bp Therefore, for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1...

...the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg...Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 ...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration ...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. This is...

...growth. See our sample data section below for positive and negative test results. Sample Data In all cases...bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, ...on the number of plates you’d like to pour. For example: Because we’d like to make 20 plates, and our plates...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 ... any weird and wonderful organisms. While your samples are sterilizing in the autoclave, you should prepare... you should create a 1000x stock solution. For example: If you’ll be preparing plates with a final concentration...

...Calculate your DNA sample concentration and purity using Addgene’s DNA Quantification Protocol....generally used as an indicator of the purity of DNA samples. These days, many labs have a NanoDrop, which is...lab. Spectrophotometer Tips Before measuring any samples, be sure to ‘blank’ the spectrophotometer using...your solvent. If using a NanoDrop to measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal... purity ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy ...accuracy of the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible... idea to re-zero any spectrophotometer between samples if they are expected to vary significantly in concentration...

...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL...fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume...Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus...follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...to 8 h and the media can be replaced with DMEM complete. However, antibiotic-containing media should not...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. Incubate...