全部

An end labeled DNA probe is incubated with a purified DNA-binding factor or with a protein extract. The unprotected DNA is then digested with DNase I such that on average every DNA molecule is c ...

Bst DNA polymerase-catalyzed radiolabeled two-step sequencing reactions (26) are modified from those presented earlier (25) by altering the absolute amounts and the relative deoxy/dideoxynucleotide ra ...

'Phenol' as used is Analar grade. Phenol should be melted at 65℃,8-hydroxyquinoline added to a final concentration of 0.1%, and equilibrated three times with an equal volume of 1M Tris.HCl, pH 7.0. The final Tris wash is replaced with TE (10mM Tris, 1mM EDTA, pH8.0) and the phenol stored in the dark at 4℃. 8-hydroxyquinoline is added to prevent oxidation of the phenol and to act as an inhibitor of RNases. 'Chloroform' as used is a water saturated 24:1(v/v) mixture of chloroform and isoamyl alco

Protocol For the original protocol describing CGH see the article by Kallioniemi et al . For an example of how this technique has been utilized by the NCI Prostate Cancer Working Group see the section on Cell Lines.DNA RNAProteomicDNA sequence copy number changes throughout the genome in a single ...

PLATE 溶液： 40 ％聚乙二醇（ PEG ，分子量 3350 ； Sigma P 3640 ） 0.1mol/L 醋酸锂

Wear gloves throughout and work in radiation area. Monitor area before and after use. Mix the following in an eppendorf tube: 1. 0.5 microgram oligonucleotide dissolved in H2O. 2. 3 microliters 10x kinase buffer. 3. 2 microliters 32 P ATP from ICN (>5000 ci/mmole). 4. H2O so that the final volume is 30 microliters. Add 25 units T4 polynucleotide kinase and incubate 60 min at 37 ℃. Purify labeled Oligonucleotide away from unincorporated ATP Currently, we use mini Quick Spin Oligo Columns (#1 8

Recovery of DNA from Low Melting Point Agarose Gels 1.Run digestion products on 0.7% LMP agarose gel in 1X TBE (it's nice to have at least 1ug of the fragment you want). LMP agarose is fragile; pour gel with EtBr 2.Let solidify in cold room. Be sure to overlay the gel with buffer before pulling out the comb, to prevent damage to the wells 3.Run gel in cold room, 100V 4.Cut out fragment of interest with clean razor blade and remove all excess agarose from the DNA. 5.Use long wave UV to visualize

PEG Preparation of Plasmid Plasmid isolated by this procedure can be used routinely for electrophoretic analysis, restriction endonuclease digestion and transformation of E. Coli., sequencing, PCR and most other molecular biological techniques. The procedure is a modification of the rapid alkaline lysis method of Ish-Horowitz and Burke (1981). You will need 3 basic solutions:- Solution I: 50mM glucose, 25mM Tris.Cl, pH 8, 10mM EDTA Solution II: 0.2M NaOH, 1%(w/v) SDS Solution III: 3M potassi

For every 4 mls of culture, dissolve the BAC DNA pellet in 40 µl of water. for example: Usually each BAC is grown in 20 mls LB/CM total, then is dispensed into one Autogen tube (4 mls in each of the 5 tubes). After miniprep, add 40 µl of water to each tube (200 µl total for each BAC). Vortex the Autogen tube and let sit for at least 0.5 hour. Then pool the 5 samples into one for each BAC. check the BAC DNA for quality and quantity by digesting 5 µl of the DNA in a 20 µl reaction: 5.0 µl DNA 2.0

This is a rapid method for chemical DNA sequencing which is commonly used as ladder for footprinting reactions or for sequencing of short DNA oligonucleotides. Reference: Bencini et al. (1984) Biotechniques 2: 4-5. Steve Hahn/Hahn Lab The method below works well for Sequencing of DNA of greater than ~40 bp. Typically, about 150 bases of sequence can be read from analysis on a 6-8% urea acrylamide gel. For sequencing of short oligonucleotides, the reaction times should be increased as noted below

DNA absorbs ultraviolet light due to its highly conjugated nature. DNA may thus be easily quantitated in a UV spectrometer. Typically 1 OD260 (i.e. a solution having an absorbance of one unit at 260 n ...

This technique can be used to isolate overlapping DNA fragments starting with a previously cloned DNA fragment that maps near a gene of interest (dark red). The walk is continued until a clone containing the desired gene is identified. In this example, the chromosomal DNA fragments are cloned in λ pha ...

The following guidelines should be taken into account when designing modified oligonucleotides. 1.Sequence Length - SYNTHEGEN can synthesize oligonucleotides from 5 to 110 bases in length. Most seque ...

alimama_pid="mm_10043573_137377_1822161"; alimama_titlecolor="FF9900"; alimama_descolor ="552c55"; alimama_bgcolor="f7f7f7"; alimama_bordercolor="f7f ...

16 µl BigDye Terminator Ready Reaction Mix 1 µg BAC DNA (determined from gel) 10 pmol primer water to 40 µl Heat tubes at 95℃ for 5 min., then perform 30 cycles of: 95℃ x 30 sec 55℃ x 10 sec 60℃ x 4 min Run BACs on a 377 and load the entire sample after clean up. If you use a 3700, the loading would be very different. This IS the double recipe. Normally a reaction is only 20 µl. For sequencing we use ABIs "BigDye Terminator Cycle Sequencing Ready Reaction Kits v2.0 with AmpliTaq DNA Polymerase",

alimama_pid="mm_10043573_137377_1822161"; alimama_titlecolor="FF9900"; alimama_descolor ="552c55"; alimama_bgcolor="f7f7f7"; alimama_bordercolor="f7f ...

A simple reversible way to a stop restriction reaction is by adding EDTA which chelates Mg2+ thereby preventing catalysis. If further manipulations of the digested DNA are to be performed the restric ...

I have been using an alternative to wedge gels that saves acrylamide, cuts gel drying time to 20 min and gives as good or better band squashing at the bottom of the gel. It was published in BioTechniques about 3 or 4 years ago (email me if you want the reference). The method goes as such: Run the gel with 0.5X TBE in the top tank and normal 1 X TBE in the bottom tank. Just after the last (or in the case of one load - the only) load has run into the gel, put a half volume of 3 M sodium acetate in

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide. 2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 ml of 50:50 DE-52 ...

This protocol is intended to make shotgun libraries from BACS that are to be fully sequenced and assembled. To minimize chimeric clones an adaptor method is used. There are simpler protocols if just r ...