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Footprinting Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples: hormone-receptor complexes that bind to their hormone response elements transcription factors that bind eukaryotic operators, enhancers, and silencers the lac repressor that shuts down the lac operon in E. coli [Discussion of the lac operon] How, for example, does one determine the DNA sequence to which the lac repressor binds? The procedure: Clon

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Wash one 0.45um Millipore nitrocellulose filter (Cat# HAWP01300) in 1ml 0.4M KOH for 1 min; wash in 3ml dH2 O for >1min. Assemble filter apparatus: white plastic bottom support/funnel nitrocellulos ...

限制酶在各种NEBuffer 中的活性 (NEBuffer Activity Chart for Restriction Enzymes) 对应每一种限制性内切酶，New England Biolabs 均提供彩色盖子的10X NEBuffer，以保证100%活性。下面表格中列出每一种酶及其随酶提供的最适宜的NEBuffer。同时也列出每种酶在四种不同的NE ...

DNaseI Footprintint Solutions 10X Binding Buffer 200 mM Tris 8.0 200 m l 1M Tris pH 8.0 500 mM NaCl 100 m l 5M NaCl 10 mM EDTA 20 m l 0.5 M EDTA pH 8.0 680 m l Q store at room temperature DNaseI Dilut ...

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TE solution 10 mM Tris (pH to 7.5) 1 mM EDTA (pH to 8.0 to dissolve) 2.Frozen agarose gel piece containing the desired DNA fragment Supplies: 1.Micropipetter and tips 2.Microcentrifuge and tubes 3.Spatula 4.PCR machine and the 600-ul tubes for use in the machine 5.Ultrafree-MC(R) filter units, 0.45 μm Procedures: 1.Quick thaw the gel piece in a 600-μl tube using the PCR machine at 37 ℃ 2.Macerate the gel piece with a spatula. 3.Transfer into the sample cup of the filter unit. 4.Spin th

Sephadex G-50 spun column purification Spin column purification can be used to change buffers without a concomitant change in solution volume to remove protein contaminants or to purify plasmid DNA s ...

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Pouring the Gel Outline: Pouring this big & thin 6% acrylamide gel ("Mother of all gels") is quite a challenge and probably the reason why smart whimps by them ready to use. Supplies & ...

This protocol works well from a 5 ml starting culture or a 1 ml starting culture (adjust volume accordingly). Solutions 2X YT Media 16 g tryptone 10 g yeast extract 5 g NaCl 1 ml 1N NaOH up to 1 liter with Q Ampicillin Stock (1000X) 0.15 g ampicillin 1 ml Q can be stored at 4℃ for several weeks Tetracycline Stock (1000X) 15 mg tetracycline 500 m l EtOH 500 m l Q vortex to dissolve and store at 4℃ Kanamycin Stock (1000X) 50 mg kanamycin 1 ml Q can be stored at 4℃ for several weeks 20% PEG 8000/ 2

1. Add 400 μl of TE buffer then 400 μl of 1-butanol to the oligonucleotide glass vial. 2. Vortex well, then spin down in tabletop centrifuge for 10 minutes at ~2,000. rpm¹s. 3. Remove top, butanol layer with a sterile pipette tip and discard. 4. Add another 400 μl of 1-butanol. 5. Vortex well, then spin down as above in tabletop centrifuge. 6. Remove top, butanol layer and discard. 7. Transfer aqueous phase into a new 1.5 ml tube. 8. Dry in a speed vac for about 5 minutes to remove all of the b

1. Pick single colony and inoculate 250 ml of LB broth containing 100 m g/l ampicillin or appropriate antibiotic. Shake at 250 RPM overnight.2. Centrifuge cells in a Sovall GSA (250 ml)or SLA-3000 (500 ml) rotor at 5 k × g for 10 minutes.3. Resuspend cell pellet in 5 ml of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8) by ...

Restriction digests consist of: 15.75 ml ddH2 O 1 µl 10 X buffer B (Boehringer Mannheim) 0.25 µl HindIII (40 U/µl) 3 µl DNA Set up digestions in 96 well plates. Incubate at 37℃ for 4.5 hours. This can be done in a thermocycler. After digestion, a brief centrifugation will collect DNA at the bottom of wells. Seal plates with foil tape and store at 4℃ if necessary. Prepare 1% agarose gels in 1X TAE: Cool molten agarose to 46℃ in a water bath with occasional stirring. Pour into 20X25 cm UV tra

Materials: RPMI 1640 medium fetal calf serum (FCS) 20% Colcemid (e.g. Boehringer Mannheim cell biology reagents Best.-Nr. 295892) cell cuture flask Phythemaglutinin PHA-L (Seromed M 5030) CO2 ce ...

Introduction This manual is a compilation of many of the everyday methods used in the average molecular biology laboratory with emphasis on the techniques for large scale DNA sequencing protocols and ...

This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher than Protocol D.1, there is considerable chromosomal DNA, RNA and protein contamination. Solutions Sucrose/Tris 25% sucrose 25 g sucrose 50 mM Tris pH 8.0 5 ml 1M Tris pH 8.0 up to 100 ml with Q store at room temperature Triton Lysing Mix 5% Triton X-100 5 ml Triton X-100 5% sucrose 5 g sucrose 50 mM Tris pH 7.5 5 ml 1M Tris pH 7.5 50 mM EDTA 10 ml 0.5 M EDTA pH 8.

Solutions Gel Stocks Diluent 5X Buffer 25% Acrylamide 209 g Urea 209 g Urea 209 g Urea up to 500 ml Q 250 ml 10X TBE 120.8 g Acrylamide up to 500 ml Q 4.1 g BIS up to 500 ml Q 2.5 M NH4 OAc 19.2 g NH4 OAc up to 100 ml Q Formamide Dye 9 ml deionized formamide 500 m l 10X TBE 500 m l 0.2% bromophenyl blue and 0.2% xylene cyanol Other Reagents Needed: 0.22 m M disposable syringe filters, short wave UV source, intensifying screen for UV shadowing Procedure • Pour a 20% denaturing gel: 12 ml 5X Buffe

T-RFLP（末端限制性片段长度多态性）该技术在应用的过程中，肯定需要在实验条件上不断进行改进，而这种改进的好坏自然而然需要实验结果的验证。 V. Grüntzig在2002年做了该工作的一部分，结果认为，在限制性酶切时，很有必要去除其中影响DNA的酶切过程，并且实验证明了具体的酶切时间。 具有说服力的结果如下： 1、T-RFLP出数据的速度快，不过只是具有半定量性； 2、DNA片断的分 ...

This protocol was designed to generate directionally end-labeled probes for DNaseI footprinting but it can be used for any application that requires end-labeled DNA probes. Solutions 10 mM dNTP Stocks Thaw100 mM stocks (NEB or Boehringer Mannheim) on ice and dilute 10-fold in Q. store small (10-20 ml) aliquotes at -80 degrees and thaw on ice just prior to use 10 X Klenow Buffer 0.5 M Tris 7.5 500 ml 1 M Tris 7.5 0.1 M MgCl2 100 ml 1 M MgCl2 10 mM DTT 100 ml 0.1 M DTT 0.5 mg/ml BSA 50 ml 10 mg/ml