...slight melting of the 5 ́-terminus during the reaction. This “peeling back” of the 5 ́-phosphorylated base...anyway? Let’s talk about it. DNA ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated... in vitro, Taq DNA ligase will ligate only nicks (5-8). Taq ligase is a NAD+-dependent DNA ligase from... the downstream side (the base pair providing the 5 ́-phosphate to the ligation). The structural/mechanistic...PMID: 19329793. PubMed Central PMCID: PMC2719376. 5. Barany, Francis. "Genetic disease detection and DNA...Veronique Bailly, and Walter G. Verly. "Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-nucleotide...reaction in a PCR world." PCR Methods Appl 1.1 (1991): 5-16. PubMed PMID: 1842922. 13. Wiedmann, M., et al....

...12 (5 min., 37 ºC -> 5 min., 16 ºC) x 30 (5 μl) 245 99.5% 48,900 489,000 24 (5 min., 37 ºC -> 5 min....amplicon form (shown above) are flanked by BsaI sites in an orientation that results in 5’ four base overhangs...similar Type IIS restriction enzyme cutting results in 5’ four base overhangs complementary to the overhangs... and accurate single-insert assembly Intermediate 5- or 12-fragment assembly More complex 24-fragment ...frequencies as the antibiotic selection markers. The 5-, 12- and 24-assembly systems were based on the ability...pGGA destination plasmids. A final validation of the 5-, 12- or 24-fragment test systems was performed by...assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases in the number of transformants...

...restriction site (GAATTC) to the 5’ end of this primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC...consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme ...Therefore, our Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds..., resulting in a final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse...final 18 bases of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC...restriction enzyme digestion. This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with...put the sequence we chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this...

... flanked by two 8 bp cutting enzymes: NotI at the 5' end and AscI at the 3' end. These inserts can then...important in cellular recognition processes. Laboratories interested in these approaches have created ... and secreted proteins Mouse sperm Mouse egg Laboratories using AVEXIS: Christian Sollner, Max Planck ...

...Species Anti-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] Integrin alpha-5,Integrin beta-1 Human, Mouse...Rabbit Anti-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.4] Integrin alpha-5,Integrin beta-1 Human, Mouse...-Integrin alpha V beta 5 [IPI-ITGAV/ITGB5.9] Integrin alpha-V,Integrin beta-5 Human, Mouse IgG1 Rabbit... IgG1 Rabbit Anti-Integrin alpha 5 [IPI-MFR5] Integrin alpha-5 Mouse IgG1 Rabbit Anti-Integrin ... IgG2a Mouse Anti-Integrin alpha 5 [IPI-MFR5] Integrin alpha-5 Mouse IgG2a  Rat   Targeting integrins...Human IgG1 Rabbit Anti-Integrin alpha M [IPI-CBRM1/5] Integrin alpha-M Human IgG1 Rabbit Anti-Integrin...Human IgG2a Rat Anti-Integrin alpha M [IPI-CBRM1/5] Integrin alpha-M Human IgG2a Mouse Anti-Integrin...

...modular inserts that span the human adenovirus serotype 5 (HAdV5) genome that can be assembled in a one-tube...information about: Adenoviral components Adenoviral plasmid construction Frequently asked questions about adenovirus...

...predictable than NHEJ. As seen in the figure above, short (5-25 bp) regions of homology flanking a double-stranded...end-joining, requires only very small homology regions (5-25 bp) for repair, making it easier to construct targeting...deletions common. In contrast, MMEJ uses regions with 5-25 bp of microhomology flanking a DSB to repair DNA...Briefly, the PITCh vector should be constructed with 5’ and 3’ microhomology to the insertion locus flanking...side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies in the genomic locus. The ...stranded breaks allow for two sets of microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette...Generate microhomologies in the PITCh vector ~20 bp 5’ and 3’ microhomologies are added to the GFP-Puro ...

...retroviral transfer plasmids that have inserts. ID Plasmid 5'LTR Description PI 1764 pBABE-puro MoMLV For transgene... information about: γ-Retroviral packaging systems Frequently asked questions (FAQ) about retrovirus Popular...Learn about retroviral plasmids at Addgene, including popular packaging, envelope, and transfer plasmids...

...share links to research that isn’t behind paywalls. 5. The #1 (re)tweeted science domain by scientists was...domains more often than other scientists   Figure 5: Portion of Tweets with Scientific Domains, Categorized...Tools & Tips: Online Social Networking for Scientists 5 Reasons to Use Reddit for Science Communication 10...use Twitter professionally to network, learn more about alternative careers for scientists, and share cool....” This paper addresses the following questions about scientists on Twitter: who are they? What do they...

...this is a great resource to have in your pocket.   5. +AMINO- by iGreenEarth AMINO is a very simple app...saved and cannot be deleted, so there is no worry about misplacing your results or losing that lab notebook...enzyme, or name to discover all the information about that restriction enzyme or share it via email with... this app. However, if you get quizzed randomly about a hazardous chemical and can’t find your lab’s MSDS... new researchers or students beginning to learn about biochemistry. The least this app will do is allow...

...there have been varied reports of titer losses from 5% to 50% per freeze thaw cycle (Krajden et al., 1999...µm or 0.22 µm filter) or by a centrifugation step (5 minutes at 300-500 g). After removing packaging cell...medium with half the volume typically used (e.g., use 5 mL medium on a 10 cm plate). By reducing the culture...efficiency by 1.5-fold (Stockschläder et al., 1999). 5. Check your packaging cells for infection If your ...PubMed PMID: 10325307. PubMed Central PMCID: PMC84922. 5. Ugai, Hideyo, et al. "Stability of a recombinant ...grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of your virus, perform a diagnostic...Additional Resources on the Addgene Blog Learn All about Adenovirus Associated Virus New to Virus? Check ...

...topoisomerase I as this enzyme recognizes the DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this...and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different DNA strand comes ...together the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place your reaction... you should reduce your incubation time on ice to 5 minutes (incubating the full 30 minutes will not significantly...significantly improve the transformation efficiency).  5. Select and Analyze 10 White or Light Blue Colonies...restriction digest, or sequencing. Pro tips Do not add 5’ phosphates to your PCR primers; you need that free...temperature, it is not recommended that you exceed the 5 minute time limit (lower transformation efficiencies...

...they have a chimeric 5'LTR. Second-generation transfer plasmids have a wildtype 5'LTR, which requires ...have a chimeric 5'LTR that includes a CMV or RSV promoter as well as a portion of the 5'LTR. Including ...Including a chimeric 5'LTR removes the requirement for the HIV Tat protein, thus decreasing the probability of...3rd generation packaging systems have modified the 5’ LTR of the transfer plasmid to eliminate the need...with a 2nd-generation system because the wildtype 5'LTR promoter requires Tat to function. Third-generation...Plasmids 101 Learn about genome-wide screening with CRISPR and Lentivirus Read about the ClonTracr Library... the proper cell line is used; several common laboratory cell lines including 293 were immortalized with...

... kinase C Protor Also known as PRR5; proline rich 5 PTEN Phosphatase and tensin homolog Rictor RPTOR independent... the cell and regulates the switch from anabolic to catabolic processes. Active mTORC1 promotes protein...mTOR pathway. enhanced signaling of mTOR, a key metabolic regulator, correlates with poor prognosis in many...ammalian t arget o f r apamycin (mTOR) is a key metabolic regulator controlling cell growth and proliferation...energy-intensive processes. mTORC1 also blocks catabolic processes including autophagy and lysosome biogenesis...

...Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment of the 5' nontranslated region of encephalomyocarditis...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event occurs...to spark translation initiation independent of the 5’ cap.  IRES elements are very useful and commonly ...S N P G P * (GSG) residues can be added to the 5' end of the peptide to improve cleavage efficiency...of an IRES fluorescent reporter for easy detection.5  Just released: Addgene's Plasmids 101 eBook And now.../journal.pone.0018556. Epub 2011 Apr 29.) PubMed. 5. Scalable signaling mediated by T cell antigen receptor-CD3...genes above with your gene or genes of interest. Some of the plasmids listed in the table above (and their...

...Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 5 P1146-1159. https... demethylases; they are dioxygenases that oxidize 5-methylcytosine to create rare DNA bases that are recognized... due to gRNA design restrictions, anything within 5 base pairs should work well, and up to 10 base pairs... 9, 597. https://doi.org/10.1038/s41467-017-02708-5 Hrit J, Goodrich L, Li C, et al. (2018) OGT binds ...King JR, Song H, Sweatt JD (2013) TET1 Controls CNS 5-Methylcytosine Hydroxylation, Active DNA Demethylation...Daniel M. Sapozhnikov, a graduate student in the laboratory of Dr. Moshe Szyf at McGill University. Some ...expression, confounding any potential conclusions about the role of methylation (Hrit et al. 2018, Kaas ...

...LbCpf1) 5' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV ... of a guide RNA provided that target is upstream (5') of a protospacer adjacent motif (PAM) Cas9 endonuclease...that you know about all the different components of CRISPR, It's time to start thinking about how you can...Use the following flow chart to get you thinking about the types of experiments you're planning on doing...

...am and Joanne didn’t have to start answering until 5 pm). The ability to see the questions first makes ...Joanne enough for all the work she put into this. 5. Reddit forums are evergreen! With all the great questions...not too worried about selling yourself and really do just want to get information about yourself and your...where users can participate in forum discussions about a wide variety of topics. Individual forums are ...where users can ask designated scientists anything about a particular topic. Given Joanne’s expertise in... is well moderated. One of our original worries about sharing science advice on Reddit was that we’d be...answering questions. r/Science has pretty strict rules about not being too sales-y in posts which falls in line...

... audience questions and an audience member asks a 5 minute question that is really a speech? You have ...event is what is important. Choose 4 panelists (maybe 5) and no more. Too many panelists will negatively affect...going to be good at talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of... are nice if you can afford them). I am talking about making sure the audience is interested, educated...of the stories or topics you know they can speak about. There should be some spontaneity, but having a ...location and parking information and remind them about the event as it approaches. Room set-up and logistics... make or break the discussion section. And what about those audience members that want to take over? It...

...without worry, as I had never worked the traditional “9-5” job. Prior to this, the only work experience I had...Science Communication function at Addgene. Working a 9-5 As it turned out, Addgene uses something called “core...accustomed to the routine, I realized that the “9-5” was not as scary as it seemed. The transition into...intimidating to me, as I had a hard time thinking about how I could fill this time. I was relieved to find...attend to other commitments, without feeling guilty about missing work. As long as my work was finished on...