... using the streak plate technique. Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs...successful streak plate, first the bacteria are inoculated over a quadrant of an agar plate. Then, a stick...streak plate skills? Find the chromoprotein expressing plasmids here, and check out our streak plate protocol...he knew it would be a perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent...transformation efficiency.  Aside from the Streak Plate Challenge, Westenberg’s course includes Winogradsky...

...developing a moderate throughput functional genomic platform to annotate genetic perturbations that may play...therapeutic interventions for cancer. This screening platform is an incredible boost to the cancer research ...

...humans. Then, one of them heard about our online platform, called Crowdfight, and filed a request for help...Crowdfight-COVID19 Crowdfight was born as a collaborative platform to help researchers working on COVID-19. Its origins...grew and became Crowdfight COVID-19, an online platform to connect researchers working on COVID-19 with... designers and other professionals, joined the platform; while they are a minority, their help has been... to push certain projects. The idea behind the platform was simple: any person working on COVID-19 could...19 One year on, our biggest lesson running the platform has been the realization that there is a great...those involved in COVID-19 research, but our new platform Crowdfight is also open to other topics.  In this...

...transfection into plates containing selection agent at different densities, then dispose of the plates that do ...) to transition from a 48-well cell plate to a 96-well assay plate. We generally freeze at least 2 ampules...cloning rings, the remainder of the colonies on the plate dried out and died. The alternative to cloning rings... rings, dilution cloning into 96-well plates, is extremely time- and resource-consumptive, since only ...cell can give rise to single clones, and thus many plates must be examined for single clones and then handled...12-48 individual colonies by (carefully) holding plates up to the light, where they appear as opaque dots... achieved by splitting transfected cells into 3 plates at different densities for selection). A 2% agarose...

...requires a template of high homology to the endogenous target, yet to facilitate the repair, the template should...protocol that amplifies the entire plasmid template. The parent template is removed using a methylation-dependent...resistant to Cas9 cleavage. If a plasmid contains the template, site-directed mutagenesis can be used to mutate...plasmids. PCR primers (green) amplify the plasmid template (blue), and introduce a restriction site “A*” ...the targeted region. Upon PCR amplification, the template is removed by DpnI restriction digestion leaving...circle rather than a linear product (see figure). Template A high purity plasmid prep significantly increases...you may want to try different concentrations of template (e.g. 0.1-1.0 ng/μl). Smaller plasmids (~3 kb)...

...modifications exist in the template DNA. There are several unique HDR mechanisms, but all templated-repair pathways...along the way. HDR uses homologous sequence as a template for repair and is thus the more accurate mechanism...process is generally error-free provided the DNA template used for repair is identical to the original DNA...one strand of the homologous DNA donor and can template repair off the other strand; this results in the...mechanism that notably does not require a donor template and thus won’t be covered in detail here. Classical...pathway, the 3’ ends invade an intact homologous template to serve as a primer for DNA repair synthesis,...portion of the invasive strand is displaced from the template and returned to the processed end of the non-invading...

... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...from low efficiency when a standard dsDNA repair template is used. The original design for creating a conditional...targeting two intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand...ameliorate this problem. Briefly, you design your donor template to include a T7 promoter upstream of the left ...vitro transcription, and use the IVT product as a template for reverse transcription to create your ssDNA...promoter will not be included in the final ssDNA template. Although effective, ivTRT may be more challenging...tested by Quadros et al. When delivering the ssDNA template and CRISPR machinery, Quadros et al. recommend...

...and plate transformants for isolated colonies (150-200 per plate) across 20 fresh selective plates. Step...gently down onto a plate of colonies. Mark the orientation of each membrane on its plate before moving on...on. Then peel each membrane off the plate (saving the plate so you can regrow the lifted colonies overnight...outcompeted by others in liquid culture, dilute and plate the complete library of transformants for single...single colonies (this usually requires 10-20 plates) (see box below). This initial transformation should be...over and place it colony-side up onto a fresh LB plate containing an appropriate inducing agent (0.4 mM...protein induction is done, peel the membranes off the plates and place each onto a lysis solution-soaked filter...

...the template. The opposite is true if the sense strand is nicked for template formation: template will...low primer-to-template ratio is used to promote annealing of one primer to each template. The PCR product...Error-prone PCR: This method amplifies the wild-type template with a “sloppy” version of PCR that results in...). It uses a uracil labeled wild-type ssDNA as template for a site-directed mutagenesis PCR that uses ...strand (step 2 and 3). Then the uracil-labeled template strand is degraded by uracil DNA glycosylase (...present on both strands of the plasmid. The ssDNA template is generated by transforming a backbone phagemid...days to generate the phage-derived uracil labeled template, this method requires further DNA shuffling to...

...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...

...). 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 ...

...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...

...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 ...

... 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...