...FBS can be purchased already head inactivated or it can be inactivated in the lab by heating to 56 °C for...glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Low serum medium such as Opti-MEM or Opti-Pro...bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C....

...recommended temperature should kill most potential contaminants, certain types of spores will stubbornly survive...For example: If you’ll be preparing plates with a final concentration of 100 ug/mL ampicillin, you should...growth of only the resistant strain. If you get this final result, your plates are ready for use or storage...any temperature and should always check for contamination prior to use. Negative Result 1: Both Strains...

...by restriction digest of existing sites in the original vector. Designing oligos To add NdeI, PacI, AscI... and CAGCT - 5' to the bottom oligo, making our final oligos 34 bp each: Top oligo: 5' - AATTC CATATG ...this would destroy the EcoRI and SalI sites in the final vector. Order the following oligos from your favorite...

... material you’re weighing and prevent cross-contamination with other substances. They also help you transfer...sterile (if necessary), to prevent any potential contamination. Scoop out the material you are weighing onto...much material, don’t put any extra back into the original container. Dispose of the excess material into...

...Background Information The final step in the construction of a recombinant plasmid is connecting the insert...vector Insert or water + Any colonies indicate contamination of intact plasmid in ligation or transformation...

...DNA segments you will need to join to create your final plasmid. Adjacent segments should have identical...digest . Sequence the important regions of your final plasmid, particularly the seams between the assembled...especially useful for introducing promoters, terminators, and other short sequences into the assembly ...

...where the image is further magnified before it finally reaches your eyes. These lenses determine the magnification...lens provides 10x magnification. To determine the final magnification of your image, multiply the magnification...sample. To ensure that your sample is adequately illuminated, look through the eyepiece. Adjust the power ...

...Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any protruding...minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. Run 2 μL on a gel...

...band to cut without having DNA in other lanes contaminating your sample. To accomplish this, it is best ...each buffer to add during the DNA isolation step. Finally, you will want to isolate the DNA from the gel....

...produce antibodies Antibody Purification Purify recombinant antibodies using Protein A or Protein G columns... Stain Determine purity and concentration of recombinant antibodies Watch the Video! Western Blot Separate...

...of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer...mins. Optional : Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually...

...higher competency cells. Additionally, if your final product is going to be very large (>10kb) you might...ligation reaction. Isolate the Finished Plasmid Finally, you will need to pick individual bacterial colonies...

... Antibody Guide Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Introduction Western...Assay Kit or other preferred method for protein determination. Prepare a 50:1 Reagent A to Reagent B dilution...

...equipment, and reagents RNase-free (Use an RNase decontamination solution, such as RNaseZap® or RNase AWAY®,...microcentrifuge tubes, 4 mL polypropylene tubes RNase decontamination solution like RNase AWAY® or RNaseZAP® Isopropanol...

... primers) or too short. Short primers produce inaccurate, nonspecific DNA amplification product, and long...

...) control of DNA recombination and gene expression can be advanced by the coordinated use of fellow SSR...system. The FLP recombinase recognizes FRT sites and initiates site directed gene recombination between those...channel under the control of Cre recombinase are used in combination with mouse lines that express Cre...regulated recombinase - keeping Cre out until needed But what can be done when the recombination should ...concentrations in combination with low concentrations of vectors expressing the required recombinase. The high...under the control of Flp recombinase together with inducible Cre recombinase (CreERT2), visualizes individual...Tamoxifen injection induces Cre-ERT2 recombinase, leading to recombination of the loxP sites and thereby to...

...addgene.org and include: The sequencing result The original trace file (.ab1 file) provided by your sequencing...

...components into a destination vector in a predefined order. Finally, the destination vectors are used ...positional groups. Eight final destination vectors, consisting of all four final RVD modules in each of ...promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant terminator, and plant...Gateway compatible destination vector for a maximum of 24 elements assembled into a final transgenesis construct...light-activated receptor tyrosine kinases Receptor tyrosine kinases (RTKs) are a large class of cell-surface...determined by competing recombination events at the genetic level. Upon Cre/loxP recombination, each transgene...:1852-61. A quicker method for protein inactivation Your protein of interest does x, but it also...

...models that express recombinases like Cre or Flp in their cells of interest and recombinase-dependent AAV vectors...Causes of recombination and leaky expression While leaky expression and spontaneous recombination is a known...sets of homologous recombination sites, they hypothesized that spontaneous recombination between these sites...even begins. This spontaneous recombination persists even in recombinase deficient E. coli and under conditions...vectors are much more prone to recombination and can exhibit recombination levels as high as 1-3%. In light...the recombination rate The first step in optimizing DIO/FLEx vectors is to know the recombination rate...system is off-target or spurious expression of recombinase-dependent AAV vectors. In this article, we will...

...against random recombinants.   Design your targeting construct. For recombination to occur in a ... for recombination, the negative selection marker selects against improper, random recombination into ...random recombination may incorporate the negative selection marker into the genome. This final product...After recombination has occurred, you'll first remove the resistance marker using Cre recombinase. Since...are 3 loxP sites, recombination can occur in multiple ways. The desired recombination event will remove...strategies to investigate a gene's function is to inactivate, or "knockout", the gene by replacing it or disrupting...yeast, mice, or Drosophila through homologous recombination. The concept is simple: deliver a template with...