...strands of the template DNA first have to be separated. This occurs by heating the dsDNA template to a point...categories of multiplex PCR:  Single template PCR reaction - one template is amplified using several forward... reverse primer sets.  Multiple template PCR - multiple templates with different primer pairs that align...polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique single stranded... primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...To determine the concentration of the starting template DNA the fluorescent signal throughout the reaction...to determine the concentration of the original template DNA in the sample.  Addgene currently uses ddPCR...

...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits to only four...be obtained in eight days. The only animals on a plate, other than the parental animal, are rescued progeny...optimize a range of editing parameters: oligo repair templates with homology arms of 35-80 bp, and DNA double-strand...CRISPR whether it be by improved gRNA or repair template design, delivery, screening, and more. Here are...some highlights from the past few years:  Repair template design The Meyer lab meticulously characterized...guidelines for designing single-stranded repair templates (Farboud et al., 2019).   The Mello lab also reported...

...researcher Growth of strain USO hisB- pyrF- rpoZ- on plates lacking histidine and containing 3-AT Scot Wolfe...USO hisB- pyrF- rpoZ- , this promotes growth on plates lacking His and containing 3-AT. FLIParaF.Ec-250n...

...). A plate with only white colonies is very suspicious! Refrigerate your plates: Placing plates at 4C...α-complementation (cell B). As shown on the representative plate to the right, colonies with an insert-containing...pigment from x-gal (included in the transformation plate medium), turning the bacterial colony blue. We should...backbone plasmid without insert. All colonies on this plate should be blue, indicating that your IPTG and x-... rush the process: It is important to give your plates enough time for any intact β-galactosidase to be... and white colonies. Take care in making your plates: X-gal is light and temperature sensitive and needs...after autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient...

...colonies on the backbone alone plate and many colonies on the backbone + insert plate (or at least more colonies...backbone alone plate). Unsuccessful ligations will usually result in few colonies on both plates (unsuccessful...), in a vector alone plate with many more colonies than the vector + insert plate (unsuccessful 2), or... each plate (unsuccessful 3).     If you have a high number of colonies on your backbone plate (greater...transformation worked. You should also verify that you are plating on the appropriate antibiotic and try varying the...the number of background colonies on your control plate (the more background, the more colonies you will...

...involved “plates,” and our scientists and lab technicians always knew from context what a “plate” was. Our...these plates now” to a fellow team member. But, at Addgene, we use seven different types of “plates” (Fig...barcode for your plate, you will get in trouble if you don’t qualify what kind of “plate” you mean. Without...to know what kind of plate they were talking about during the meetings. And “plate” certainly wasn’t the...information Prescribing an official name to all of our plates, tubes, and racks meant we had fewer errors in ...

...wells plates onto agar q-trays where the samples must be spread much farther apart before plating. With..., we can prep two 96 well plates in just under 3 hours or one 96 well plate in about an hour. Automating...too much and dying.  Also, we added a couple of platforms that fit 48 well q- trays allowing us to do up...96 tips for easy sample transfer between 96 well plates. The second pod can pick up 8 tips and position... robot is called the Qiagen BIOROBOT Universal Platform and it suits our plasmid prepping needs perfectly...on a larger scale. Using the BIOROBOT Universal Platform is ideal because it eliminates the potential of...the flask with agar in it. The agar sits on a hot plate with a magnet so that the agar doesn’t solidify ...

...process is completed in a plate format yielding anywhere from two to six plates of 96 samples per week. ...anyone who has ever worked to sequence DNA on NGS platforms will tell you, while the process has become much... process only takes about one day, even for six plates (576 individual plasmids)! These libraries are ...of this range are “failed” and removed from the plate to be prepped again. These holes are then filled... Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated...using a standard PCR cleanup kit to remove any template DNA and primers.  After performing extensive QC...

... can plate larger volumes of the outgrowth containing the transformants onto the selection plate. Figure...transformation plate can be manipulated to result in appropriate levels of colony plating densities. Ligase...protocol* (Volume of 1mL Outgrowth plated) Correct assemblies per plate Fidelity of assembly (percent correct...phenotype upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding...Figure 2 shows representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of ...and T4 DNA ligase. Efficiency of assemblies per plate using outgrowth volumes described in Figure 3, with...

..., just streak the colonies on an LB plate. You can use this plate to start liquid cultures. Lastly, you...polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze... to preparing a standard PCR reaction: combine template, primers, polymerase, and dNTPs and then incubate...released from the bacteria in order to serve as PCR template. Dealing with this and a few other colony PCR ...PCR tips are highlighted below. Preparing template: Pick a single colony with a sterile flat toothpick...background colonies on your no ligation control plate. The more background, the more colonies you will...remaining bacteria-water suspension will serve as the template for your PCR reaction. You just need to lyse the...

... Barcoded Tube Plate Freezer Rack Freezer Tubes are assigned a location within a plate that can hold ...below). Each plate has numbers and letters to give coordinates, from A1 through H12. Each plate is also barcoded... Each sample that is transformed is plated onto a petri dish plate that is also barcoded. The barcodes...rack that contains five levels that can hold five plates on each level. This rack is located in one of five...sample type. It is critical for us to know which plate, rack, and freezer each sample is located in. Otherwise...identifies the contents of each individual tube.  If a plate of samples is ever dropped, we can simply scan the...

...not hosted on the platform, they can be hosted on YouTube, Vimeo, and other platforms (such as university...appealing to many audiences. While all of these platforms were developing, Google acquired Youtube and has...quickly. Taking what I loved from each of these platforms (such as social networking, visual appeal, instant... across multiple other sites), I created a new platform for sharing video abstracts about research: www.WeShareScience.org...www.WeShareScience.org. WeShareScience is a free platform for sharing short, 3 to 5 minute videos about ...university websites). WeShareScience is a social platform that provides links to the videos, keeping the copyright...ways to share ideas derived from research. The platform uses video to complement traditional print journals...

...the blocking-only templates will compete with the blocking/intended mutation templates resulting in cells...simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide... very excited to set up a CRISPR-based editing platform in our lab. Our goal was to use the system to ... – Edit the cells a second time using a repair template that corrects the blocking mutation and again ...that the cell was only using a part of the repair template and mutations further away from the cut site were...intended mutation site are available, a repair template containing blocking and intended mutations can...can be equally mixed with another ssODN template that contains the same CRISPR/Cas-blocking mutation but...

...of cells. The antibiotic in your plate might not work. Check the plates! Streak on them an E. coli  strain...Design primers that overlap at least 24 bp with the template and if the sequence has high GC or AT content,...if you are having difficulties in amplifying a template, particularly from a large genome, you can recheck...have no colonies, check that the antibiotic in the plate correspond to the antibiotic resistance marker present...growing fine, it is probably time to prepare new plates with a fresh stock of antibiotic. Many small colonies...surrounding big colonies. You probably left your plate for too long in the incubator. The small colonies... different temperature (30°C) for the growth on plates and in liquid culture. If you don’t get any positive...

...proteins within a 96- or 384-well, flat-bottomed plate. The basic principle of an ELISA is that any antigen...works by first coating the wells of a flat bottomed plate with a capture antibody that is specific to the ...experimental sample are bound to the wells of a plate instead of an antibody. Coating the wells with a...characterize antibodies against the antigen bound to the plate. Figure 3: Overview of using Direct ELISA...protein antigen is coated onto the wells of the plate. However, instead of detecting your protein using...absorbance of each well, which is measured using a microplate reader, is directly proportional to the amount...your experiment. These are wells on the same ELISA plate that include known concentrations of your protein...

...to the template strand. Therefore, I suggest that picking a crRNA complementary to the template strand...tagging experiments by assembling csRNPs in a 96-well plate format and tested parallel tag knock-in for 96 different...single go (Figure 3). We were able to tag >30 genes/plate in the first shot with typical knock-in efficiencies...knock-in experiment were pre-assembled in a 96-well plate. Glioma stem cells were transfected in 96-well format...device; 5 days after the transfection, the entire plate was scanned for V5 tagging using an Operetta imaging... Clarke et al shows that crRNAs binding to the template strand (strand used by RNA polymerase II for transcription...e.g. convert the NGG PAM to NGC in your repair template) and use homology arms of 60 -70 bp . Purchase...

...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive colonies...for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25 μl single-tube...methylated DNA, so it will selectively destroy the template, but not newly synthesized sequences, limiting...Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while modifications...sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature (Tm... bp, such as epitope tags, design primers with template binding regions to amplify away from the insertion... frozen: just defrost and add your primers and template. Amplifying the ‘unamplifiable’ Check your amplification...

...up your plate in a way that will tell you if you have pipetting errors on the edges of the plate. Compare...variation due to technical error.  Plate readouts can be affected by plate type or tubes, sealing method, ...possible to multiplex your reference genes to save on plate space. For many cell lines and tissue types, appropriate...quantification. A consistent 5% positive error across the plate is fine; a 5% positive error on one side and a 5%...reagents can prevent your reaction from reaching the plateau stage after exponential amplification, making it...multiplexing your reference gene set can save significant plate space and may be worth the initial effort of setting...to set the channel wavelength before you run the plate.    Analyzing There are a number of methods...

... the then RNAi Platform, which has since evolved into the Genetic Perturbation Platform and encompasses...Associate Director of the Genetic Perturbation Platform at the Broad Institute and has worked with many... up on a Saturday at like 7 am to go check your plates and you’re excited to do so, you know that that...The model that the Broad was building that had Platforms with a capital ‘P’ focused on technology ... has...enjoys working on technology development under the Platform model at the Broad Institute because they are ...CRISPR Learn more about the Broad Institute and the Platform Model. ...

... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...making the cut off essentially as part of the templated DNA. Check out our blog post on increasing HDR...To make sure your donor molecule is used as the template, you need to incorporate homologous sequence to...sister chromatid is likely to exist as a repair template. To maximize HDR events, ensure cells are actively...