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Linker Ligation (with T4 ) DNA Ligase In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE buffer (5-7µl). Add 1-2µg of phosphorylated linkers in 5µl of TE buffer. Add: 10X ligation buffer 2µl, 50% PEG 4000 solution 2µl, deionized water to 20µl, T4 2u. Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. DNA Ligase Incubate the mixture for 1 hour at 22℃ . Inactivate T4 DNA Ligase by heating the reaction mixture at 65℃ for 10 minut

Instructions Modified for Simpson Lab1.PCR: Perform a standard PCR reaction using taq polymerase. (Do not use tfl as the PCR product must have a 3' adenine overhang that only taq generates.) Include an 72℃ extension step for 7 to 30 minutes at the end of the reaction to ensure that all products are full length a ...

Materials: Silica Suspension: add 2 g of silica to 15 ml of H2O wash 3x by centifugation at 2000 x g for 2 min estimate vol of silica and resuspend in 2 vol H2O Silica Wash Solution: 50 mM NaCl, 10 mM Tris 7.5, 2.5 mM EDTA, 50% Ethanol 6 M NaI

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TA Cloning I. Initial mixture 5 μl dd H2O 1 μl PCR product 1 μl ligation buffer 2 μl TA vector (add second to last) 1 μl ligase (add last) Incubate overnight at 15℃. II. Electroporation 1. Chill cuvettes on ice before starting. 2. Set out electrocompetent JS5 cells to thaw on ice. It is important to keep them on ice. 3. Dry one 100 mg/ml AMP plate for each sample. Add 40 μl of xgal and 40 μl of IPTG to each plate. 4. Add 500 μl of SOC or SOB media to each 5 ml snap cap tube. 5. Heat ligations at

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1. Remove 0.5 cm of tail into polypropylene microfuge tube (do not mince). (The tubes must have tight-fitting caps so that there are no leaks in steps 3 and 7 below.) 2. Add 0.5 ml DNA digestion buffe ...

1.grow plants in trays of 96 and leave two spots open (for the PCR controls) 2.harvest 1 to 2 young and green leaves (1cm2 /plant at rosette stage if possible). Use 96 well plates (1 or 2 ml E&K ...

1取5ml噬菌体溶液放入无菌干净的小烧杯中，缓缓加入固体NaCl，使其终浓度为1M（0.292g）充分溶解后冰浴1小时。 24℃下11000rpm离心10min，取上清液于另一个干净烧杯中，将PEG6000加入烧杯中使其终浓度为10%W/V约0.5克），磁力棒缓慢搅拌，冰水浴1.5小时，于4℃，15000rpm离心15min，弃上清。 3将沉淀溶于300μlTE（pH8.0），加等体积的酚/ ...

Materials: 1.TENS solution: 10 mM Tris (pH to 7.5) 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve) 0.1 N sodium hydroxide 0.5 % sodium dodecyl sulfate 2.3 M Sodium acetate pH 5.2 3. ...

Preparation of BAC (Bacterial Artificial Chromosome) DNA with CONCERT ™ High Purity Plasmid Purification System1 Courtesy Lisha Xu and Alice C. Young Molecular and Cell Biology Research and Deve ...

genevasans-serif" size="2"Protocol written by Minoru Toyota genevasans-serif" color="#932653" size="2"1. Materials genevasans-serif" size="2"1.1. MCA genevasans-serif" s ...

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competent cells (DH5alpha at competency of 108 cfu/µl of pUC18). The transformation procedure it self is very similar to a standard CaCl2 transformation. Place 100 µl of TB buffer in a tub ...

There are multiple variations to this protocol, but we find that this one works well in all cases we tested.Reagents: 5X EMSA Buffer: 50mM HEPES (pH 7.9)375 mM KCl12.5 mM MgCl20.5 mM EDTA5 mM DTT15% Ficoll32 P-labeled oligonucleotide probepolydI/dC: 1 mg/ml in TEBSA: 10 mg/ml in TE5% Polyacrylamide gel (30 ...

Restriction Enzyme Buffer Most enzymes can use REact buffers; however, some are made up separately. Use fresh Milli-Q water , siliconized or sterile glassware or disposable plastic ware to make the following stock solutions up fresh (discard after use), combine to make 5 ml of 10X reaction buffer ...

1.Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediat ...

The overall sequence of events is: • Titer and plate out phage • Lift plaques onto filters and prepare them for screening • Make a probe • Hybridize the probe to the filters &b ...

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Important : Extract the DNA within one week of receiving samples. Samples that have been processed should be frozen to prevent degradation. Freeze samples between use each day. 1. Add 600 m l of 50 mM NaOH to a 1.5 ml eppendorf tube and insert brush in tube. (Clip off handle of brush with wire cutters so tube can be closed. Cutters should be rinsed with EtOH between samples to prevent contamination.) 2. Vortex thoroughly to mix, for at least 10 s. 3. Heat to 95℃ for 5 min. 4. Spin briefly to poo