...purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of donor plasmid...lanes between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample...plasmid. We recommend around 100ng of total DNA in a standard ligation reaction. You ideally want a recipient...important to set up negative controls in parallel. For instance, a ligation of the recipient plasmid DNA without...instructions for your competent cells. For most standard cloning, you can transform 1-2μl of your ligation...

...containing the carbenicillin (amp) resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin... cells containing plasmids differing in their resistance genes, as one does not need to prepare separate...place of ampicillin because carbenicillin is more stable, so it is potentially more effective at selecting...

... out more protocols and videos to help you get started in the lab! Introduction For many experiments, ...precisely to create the solution you expect. Understanding how to obtain the correct amounts of materials...weighing and prevent cross-contamination with other substances. They also help you transfer the material to ...your lab’s material safety data sheets (MSDS) to understand how to properly dispose of reagents that you ...

... use it to allow the temperature to stabilize. Use water resistant markers, such as permanent markers,... out more protocols and videos to help you get started in the lab! Introduction A water bath is a piece...water bath, be sure to use a marker that is also resistant to chemicals such as ethanol. Writing on the tops...

...Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites. See ...identical, to their recognition sites. This is due to "Star Activity" and can happen for a variety of reasons...(Link opens in a new window) NEB's website about star activity . If you are digesting a large number of... of plasmids with the same enzyme(s) (for instance, in a diagnostic digest), you can create a "Master ...

... 10-013-CV L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) ...: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative such as glutaGRO) To a 500 mL bottle... a new working stock. Considerations Before You Start The health of the cell line is critical for obtaining...endonuclease negative E. coli strain such as NEB stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of ...

...the two ends of the vector together). Protocol: Standard Insert + Vector DNA Ligation Before setting up...each and their concentration. However, for most standard cloning (where the insert is smaller than the ...fine. We recommend around 100ng of total DNA in a standard ligation reaction. Use a (Link opens in a new ...of competent cells and verifies the antibiotic resistance of the plasmid Cut vector - Background due to...

...protocol describes the basic steps for fixing and staining cells in culture with a primary antibody against...15 mL conical tubes 50 mL conical tubes Before Starting Refer to the manufacturer's instructions for additional...500 µL PBS on a rocking platform. (Optional) Counterstain nuclei with 500 µL of 300 nM DAPI working solution... Review the manufacturer's instructions before starting your experiment and consider titrating your antibody...

... out more protocols and videos to help you get started in the lab! Introduction The pipette is an essential...focused on single channel pipettes. These come in standard sizes: P2, P10, P20, P200, and P1000. The table...each type of pipette. This section will help you understand how to read each display to dispense the correct...multiple “stops” on the plunger, where you feel resistance while pressing down on the pipette. Stop at the...

...shaking platform set to 120 rpm 37 °C bead bath Clamp stand and clamps Autoclave 0.1–1 mL single channel pipette...UFC903024 50 mL LoBind tubes, VWR 76289-498 Before Starting Wipe down all pipettes and equipment with 10% ...if this has not already been done. We typically start with about 250 mL of supernatant and add 250 µL ...Attach the Gravitrap column to a clamp on a clamp stand. Use scissors to cut the bottom of the Gravitrap...

...different steps in RNA extraction. Before Starting RNA is not as stable as DNA and is susceptible to degradation...microcentrifuge, see step 4 of either protocol before you start. Homogenizer Vortexer -20°C freezer -80°C freezer...Procedure Option #1 - Solution D Protocol Before starting this protocol, make sure to prepare solution D...

...protocol, the following protocol can be used as a starting point for CRISPR libraries. This protocol allows...library. This protocol assumes familiarity with standard bacterial transformation and basic knowledge of...efficiency electrocompetent cells that are suitable for unstable or recombination-prone DNA. The use of electrocompetent...down at 30 ℃ overnight. Critical Ensure at this stage that no unabsorbed media drips onto the lid. Let...

...from the publication Wang et al. (2018) . Before Starting Thaw the master mix, primers/probe mixes and samples...Heat-inactivated FBS L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) ...the dilutions. RPP30 copies should be relatively constant across samples. In the RRE example below, 2-fold...below, the concentration of RPP30 positive droplets stays relatively even across samples (green). To increase...

...DNA fragments based on size. The procedure starts with standard agarose gel electrophoresis, which separates...efficient with lower % agarose gels, so you will want to stay in the 0.7-0.8% range if possible. You will want...

... agarose using a commercially available kit or standard protocol. Anneal oligos The oligos should be resuspended...tube. Place tube in a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25...ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate an annealed oligo...

... 10-013-CV L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) ...: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or stable alternative, such as glutaGRO) To a 500 mL bottle... to 56 °C for 30 min. Considerations Before You Start The health of the target cell line is critical for...

...Get started with Gibson Assembly Cloning!...plasmids by designing primers to split an antibiotic resistance gene to effectively create an extra part, one...contiguous DNA fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein (ET SSB)...

...the SV40 origin of replication use a HEK293 line stably expressing the SV40 large T antigen. For antibody-expressing...Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr nuclear antigen (EBNA).... saline solution, Millipore Sigma A6279 Before Starting Warm the DNA and working stock of PEI-MAX to room...

... out more protocols and videos to help you get started in the lab! Introduction There are four biosafety...sometimes scientists work for a long time while standing. Wear comfortable shoes that cushion your feet...

...entire construct. Is the primer an appropriate distance from the target? The primer should be a minimum...and make sure they are justifiable peaks. For instance, in the trace file below, you can see that just...