...microscope can achieve. You achieve the highest resolution when your image is in focus, which you can control...components with science-fiction levels of detail. You can take entire courses on microscopy and still have ...you to distinguish objects within the sample. You can increase contrast by a) adjusting the aperture of...Microscope Prepared Slides (or other sample that can fit on a microscope stage) Reagents None needed Procedure...) on the microscope stage. If using a slide, you can secure it into place using the metal clips on the...Adjust the power of the light source so that you can look through the eyepiece comfortably. Then, close...knob (the larger of the two focus knobs) until you can see the edges of the aperture are in focus. Center...

...What program can I use to view my trace file? There are many free programs available that can open .ab1 ...PC), FinchTV (Mac/PC), Sequence Scanner (PC), and Chromas (PC). How can I tell if a peak is mis-called?...These primers typically anneal to the backbone and can help you verify the ends of the insert. Longer genes...that anneal to a specific region of the insert. You can find Addgene's sequencing results by clicking on ...target. Are there commercially available primers that can be used for sequencing? There are many widely used...sequencing cores have a list of common primers that can be requested for no additional charge. What primers...peaks. For instance, in the trace file below, you can see that just after base 70 there are multiple peaks...

...larger models can hold larger conical tubes. Depending on the centrifuge their speed ranges can vary, but ...and can be about bench height. They often spin at similar speeds to their smaller cousins, but can hold...generally floor sized centrifuges that can hold larger containers, but they can also rotate at much higher speeds...encounter several different types of centrifuges that can accommodate different sized containers, spin at different... tubes in place, and the control panel where you can set the time and speed needed for your experiment...balanced. Using the centrifuge in an unbalanced state can damage the centrifuge and be dangerous for the user...different protocols. Improper use of a centrifuge can damage the equipment, your samples, or you, so be...

...with your protocols. FBS can be purchased already heat-inactivated or it can be inactivated in the lab...Protocol that can be used to titer lentiviral preparations carrying a fluorescence marker. Protocols...Protocols Viral Service Introduction This protocol can be used to titer lentiviral preparations that carry.... This protocol was developed for 293T cells but can be adapted to a variety of target cell lines and ...the percentage of fluorescent cells, these cells can still be used in downstream applications. Safety ...calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.45 μm ... 4 °C. Pro-Tips Different brands and lots of FBS can promote or inhibit transfection. Test a variety of...

... for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many labs also create...cells and retransform. Bacteria on an LB agar plate can be stored at 4°C for a few weeks. However, if you...keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol...dH20. Snap top tubes are not recommended as they can open unexpectedly at -80°C. Freeze the glycerol stock...conditions, including copy number and growth temperature, can be found on your plasmid's information page. The ...Most labs store bacteria in 15-25% glycerol. You can prepare the glycerol stock the same time you prepare... on and samples stored for long periods at -80°C can lose labels stuck to tube! Reference Page Top Index...

...with your protocols. FBS can be purchased already heat inactivated or it can be inactivated in the lab...Guides Mol Bio Protocols Introduction This protocol can be used to produce lentivirus from a lentiviral vector...polyethylenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell lines ...transfection reagents. Once produced, lentivirus can be used for a variety of downstream applications ...calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.45 μm ...at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of...at -20 °C or -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After 2 months...

...with your protocols. FBS can be purchased already heat-inactivated, or it can be inactivated in the lab...which case this incubation step can be reduced to 8 h and the media can be replaced with DMEM complete....dilution can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately...This protocol can be used to titer lentiviral preparations that confer antibiotic resistance. Protocols...Protocols Viral Service Introduction This protocol can be used to titer lentivirus preparations that confer.... This protocol was developed for A549 cells but can be adapted to a variety of target cell lines and ...calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.1% Crystal...

...full-length cDNA you can find for YGOI is in a bacterial expression vector. Using subcloning, you can easily move...Bacterial Transformation Summary The following technique can be used to easily move any piece of DNA from one ...vector. If you are not sure what vector to use, you can check out our Empty Backbone Reference . Background... plasmid to another. However, the same technique can be used to move promoters, selectable markers, or...desired restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to...restriction sites to your recipient plasmid : You can modify the MCS of your recipient plasmid using Annealed-oligo...restriction enzyme buffers). If you select enzymes that can function in the same buffer, it will save you time...

... oligos. This is an option that can be added when ordering them or can be performed enzymatically later...Bacterial Transformation Summary Oligo overlap cloning can be used anytime you need to add a short stretch of...an empty vector. However, the following technique can be used just as easily to add short tags or shRNAs... AscI, and MfeI. With oligo overlap cloning, you can design a set of oligos containing your desired restriction...will design overlapping oligos that once annealed can be cloned directly into the overhangs generated by...oligo will be the reverse compliment so that they can anneal. Top oligo: 5' - CATATG TTAATTAA GGCGCGCC ...if necessary to get to 50μL. Efficient annealing can be achieved by one of two methods: Method #1 Place...

... homology region. Hairpins in this region can significantly reduce the efficiency of two homologous ends...compatibility, multiple overlapping DNA fragments can be joined in a single isothermal reaction. With the...joined fragments. Fewer steps. One tube reaction. Can combine many DNA fragments at once. Procedure Design...and B in the figures). These identical sequences can be created via PCR with primers that contain a 5′... correctly assembled antibiotic gene. This trick can also enable replacement of "inverse PCR" reactions...Otherwise, PCR purification or even the raw PCR mix can work fine in an assembly if you want to save time...from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other. Phusion High-Fidelity...

...with your protocols. FBS can be purchased already heat-inactivated, or it can be inactivated in the lab...well of a 6-well dish can be expanded into a 10 cm dish. A confluent 10 cm dish can be expanded into two...Protocols Viral Service Introduction This protocol can be used to generate stable cell lines expressing ...This protocol was established using 293T cells but can be adapted to alternative cell lines. Workflow Timeline...calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.45 μm ...Store at 4 °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection. Test a variety of... add penicillin/streptomycin to the media, as it can affect viability and transduction efficiency. Infection...

...the gel more quickly than longer ones. Thus, you can determine the approximate length of a DNA fragment...microwavable flask. See TAE Recipe . Pro-Tip TBE can be used instead of TAE, labs usually use one or the...Caution HOT! Be careful stirring, eruptive boiling can occur. Pro-Tip It is a good idea to microwave for...over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close...solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5...EtBr solution and then rinse it in water before you can image the gel. Pour the agarose into a gel tray with... bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the ...

...an amplification protocol, the following protocol can be used as a starting point for CRISPR libraries...., fitness differences, and plasmid recombination can all impact the representation of individual plasmids...the pooled library from Addgene, these protocols can be found on the Pooled Library page for the specific... libraries up to 200,000 individual plasmids and can be scaled according to the actual number. Required...Required Quality Control (QC) Pooled libraries can be challenging and expensive to ensure adequate quality,...restriction digest protocol here ). Lentiviral plasmids can recombine between their LTRs resulting in a smaller...per 25 µL electrocompetent cells) Less library DNA can be used but this dramatically increases the chances...

...with your protocols. FBS can be purchased already head inactivated or it can be inactivated in the lab... of a gene of interest in a target cell line and can be useful for short term studies of protein function...any HEK293T cell line should work. This approach can be adapted for different cell lines and different...at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of... Aliquot 50–100 µL and store at -20 °C. Aliquots can be thawed and stored at 4 °C prior to use. Thawed... the tubes at -80 °C. After thawing the solution can be stored at 4 °C for up to 2 months. After 2 months... into 500 µL of Opti-Pro SFM. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid DNA purification...

...vector backbone are known and thus this technique can be quickly used to verify your plasmid. The goal ...electrophoresis . The pattern of the fragments on the gel can indicate if the plasmid contains the expected size...insert. By selecting the appropriate enzyme(s), one can either linearize a plasmid to determine the size ...will cut your plasmid into multiple fragments, you can get a very unique pattern that will distinguish one...digesting with both and seeing both patterns you can be incredibly confident that you have the correct... has be cloned in the correct orientation - this can be done by restriction digest. Although this is never...cuts in the backbone on one side of the insert, you can get a very obvious verification of the orientation...

... for your protocols. FBS can be purchased already heat-inactivated, or it can be inactivated in the lab...Molecular Biology Reference Introduction This protocol can be used to produce AAV from one Five Chambers Cell-Stack...HI-FBS) Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of...calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 1 mg/mL ...the tubes at -80 °C. After thawing, the solution can be stored at 4 °C for up to 2 months. After 2 months...number of base pairs from all three plasmids, we can determine the total μg/bp we need to achieve a 1:...RT for 15 min. Note that longer incubation times can reduce transfection efficiency. Add the OptiMEM +...

... Water bath floats can be used to secure tubes in place and water bath weights can hold bottles in place... to incubate bottles and containers. Water baths can also be placed in walk-in refrigerators to achieve...remember that when you add the tube to the slots, it can cause the water level to rise up and flow over the...will likely involve a dial or a digital display you can set to the correct temperature. Set up your water...If you regularly use a water bath in the lab, you can leave the water in it from previous uses. However...

...of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from... 60 mm x 15 mm plates which can hold 5-10 mL of agar and on which you can individually distinguish a maximum...indicated, the antibiotic powder can be dissolved in dH 2 O. *Carbenicillin can be used in place of ampicillin...Protocols Cloning Protocols Introduction Plasmids can carry one or more antibiotic resistance genes, which...plates. Many labs use 100 mm x 15 mm plates which can hold 10 - 15 mL of agar. Autoclavable flasks Sterile...Because we’d like to make 20 plates, and our plates can hold a maximum of 10 mL, we’ll want 200 mL of media... you’re making plates without any antibiotic you can alternatively re-liquefy the agar by running it through...

...reverse-complement of this sequence so that we can successfully amplify the ORF. You can generate the reverse-complement...sites to the ends of that piece of DNA so that it can be easily cloned into a plasmid of interest. For ...It is helpful to choose restriction enzymes that can both function in the same buffer, as this will save...restriction site to improve cutting efficiency. You can generally add any 6 bases, but you should ensure ...reverse complement to get PCR amplification. We can start similarly, taking the final 18bases of the ...there is very little chance for mis-priming, so you can use a pretty wide range of annealing temperatures... competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...

...length in base pairs. Following electrophoresis, you can cut DNA bands out of the agarose gel and purify the...if possible. You will want nice crisp bands. This can be achieved by using a wider gel comb and running...the scale with an empty tube. Alternatively, you can just subtract the weight of the empty tube from the...sized bands that are running too close together you can adjust the gel percentage to get better separation...more volume than needed because several microliters can be lost in pipetting. For example, if you want to...available. Unlike the plastic tray, this will not significantly reduce the UV, but will protect the UV box from... during the DNA purification step, as many kits cannot handle more than a certain total volume of gel ...