...431098 Sodium phosphate monobasic monohydrate, Millipore Sigma 71507-250G Sodium phosphate dibasic, Millipore...sterilize 1 M of sodium phosphate buffer, pH 7.0 390 mL of sterile sodium phosphate monobasic monohydrate...binding buffer (20 mM sodium phosphate buffer, pH 7.0) 20 mL 1M sodium phosphate buffer 980 mL of deionized...compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform...RT) before use. Reagent Preparation 1 M sodium phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH ... 7.0 Autoclave or filter sterilize 1 M sodium phosphate dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO... (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to 1 month at 4...

...with a phosphatase before performing the ligation reaction (phosphatase removes the 5' phosphate and therefore...was completely digested and if phosphatase treated, that the phosphatase treatment worked. This control... ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides... vector re-circularization - most useful for phosphatase treated vector Insert or water + Any colonies...

...purity. This can be done with a spectrophotometer. A spectrophotometer uses the absorbance/transmission... instructions for the spectrophotometer specific to your lab. Spectrophotometer Tips Before measuring ...labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration...measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, ...NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary...

... Protocols Agarose Gel Electrophoresis Agarose Gel Electrophoresis You may also like... Restriction...Standard protocol for performing agarose gel electrophoresis, including tips to improve resolution and separation... Agarose Gel DNA Ligation Introduction Gel electrophoresis is the standard lab procedure for separating...pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively...protocol video below to learn how to perform gel electrophoresis. Equipment Casting tray Well combs Voltage ...solidified, place the agarose gel into the gel box (electrophoresis unit). Fill gel box with 1xTAE (or TBE) until... short DNA fragments is polyacrylamide gel electrophoresis (PAGE), which is typically used to separate...

...separated by denaturing polyacrylamide gel electrophoresis alongside serial dilutions of a standard antibody...pipette Pipette controller Microcentrifuge Electrophoresis chamber Power supply Rocking platform Fume ...the bottom of the gel. Place the gel in the electrophoresis chamber and secure it. Note: Check the manufacturer...make quantifying the gel easier. Cover the electrophoresis chamber and attach to a power supply. Run the.... Turn off the power supply and unplug the electrophoresis chamber. Remove the gel from the chamber. Use...background staining. Pro-Tip A large shift in the electrophoretic mobility of the bands (sample AR0016 in Figure...ImageJ (Link opens in a new window) or a similar photo software to determine the relative intensity of ...

...insert. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are commonly used. Follow...your restriction digest results. Equipment Electrophoresis chamber Pipetman Reagents Liquid DNA aliquot...manufacturer's instructions) Gel loading dye Electrophoresis buffer Pipet tips Procedure Select restriction...visualize the results of your digest, conduct gel electrophoresis . Tips and FAQ Restriction enzymes MUST be ...enzyme to cut the vector, you will need to use a phosphatase to prevent re-circularization of the vector if...

... the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are ...to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as...overhangs after digestion, you will need to use a phosphatase to prevent re-circularization of the recipient...treat your digested recipient plasmid with a phosphatase prior to the ligation step or prior to the gel...

...Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis Introduction Restriction enzymes are naturally...and analyze the resulting fragments by gel electrophoresis . The pattern of the fragments on the gel can...to analyze a restriction digest. Equipment Electrophoresis chamber Pipetman Pipet tips Reagents Liquid...manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total Plasmid Size -OR- Insert... tricks see the Restriction Digest and Gel Electrophoresis pages....

... the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are ...overhangs after digestion, you will need to use a phosphatase to prevent re-circularization of the vector. ... treat your digested recipient vector with a phosphatase prior to the ligation step or prior to the gel...

...gel electrophoresis, which separates DNA by their length in base pairs. Following electrophoresis, you...Restriction Digest of Plasmid DNA Agarose Gel Electrophoresis DNA Ligation Background Information Gel purification...Protocol: Gel Purification Follow the Agarose Gel Electrophoresis Protocol with the following amendments: Notes...

... Technology Protocol Agarose Gel Electrophoresis Gel electrophoresis is the standard laboratory procedure...visualization and purification. Agarose Gel Electrophoresis Protocol Purifying DNA from an Agarose Gel ...DNA fragments based on size. Following gel electrophoresis, you can cut DNA bands out of the agarose gel...

...min. 25 mM chloroquine diphosphate Dissolve 0.129 g of chloroquine diphosphate salt into 10 mL of sterile...fresh DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete,...complete, add 10 µL of 25 mM chloroquine diphosphate. Dilute 18.9 µg of DNA into 500 µL of Opti-Pro SFM. Pro-...

...If you plan to phosphatase treat your cut vector it is necessary to use 5'-phosphorylated oligos. This ...

...Information Anatomy of a Pipette This front view photo of a P1000 pipette shows the plunger at the top,... pipette tip off of the pipette. This side view photo of a P1000 pipette shows the volume adjustment ring...ready to begin using the pipette. To the right is a photo of how you might hold a pipette. Without moving ...

...of each well at 450 nm on the spectrophotometer. Use the spectrophotometer to generate a standard curve...an automated ELISA plate washer. Equipment Spectrophotometer compatible with 96-well plates 1–10 µL single...

...cloning scars"). A “stuffer” sequence allows for electrophoretic separation of linearized vector from the reaction...linearized vector from the reaction mixture by gel electrophoresis followed by gel purification . The cut vector...

...Quantification Measure DNA concentration with a spectrophotometer Restriction Digests Cut and analyze plasmid...plasmid DNA with restriction enzymes Agarose Gel Electrophoresis Separate DNA by size on an agarose gel Watch...

...product can be verified based on size by gel electrophoresis . Protocol Video Watch the protocol video below...rate of the DNA polymerase. Deoxynucleoside Triphosphates (dNTPs): Are the building blocks added one at...

...the quality of your RNA sample(s) using a spectrophotometer (such as a Nanodrop), agarose gel, or bioanalyzer...

...complex in the transduced cells. hPGK Human phosphoglycerate kinase promoter drives expression of puromycin...