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(adapted from Bruce A.RoeDepartment of Chemistry and BiochemistryThe University of OklahomaNormanOklahoma 73019 broe@ou.edu) Phenol extraction is a common technique used to purify a DNA sample (1).Typ ...

Materials: 1.TE solution o10 mM Tris (pH to 7.5) o1 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 ...

酶 最适温度 37℃%活性 Apa I 25℃ 10undefined ApeK I 75℃ n ...

1.O/N (5ml E.coli in LB-->1ml into 200ml LB/37℃ 2.Grow 2-3H to A660 ~0.3-0.4 3.Chill cells 10'/ice Spin down 10'5K4℃ 4.Wash 1X 200ml 10% glycerol (sterile)40℃; Spin down 10'5K4℃ 5.Wash 1X 40 ml 10% ...

I am trying to digest mouse genomic DNA for a southern blot. When digested with either HIndIII or NdeI I get a nice smear as expected. But when I digest with either speI or BglII (neither one of which ...

Isolation of DNA from Agarose Gels (Paper Slurry Method) This procedure isolates DNA from agarose gels by filtration through a filter-paper column.The column is made in a 500 µL tube from a slur ...

This procedure was originally developed for Listeria monocytogenes but has worked well with other Gram+ bacteria we've tried. 1.Pellet cells from 10 ml overnight cultures in BHI or LB and wash in 5 ml ...

1.Pour a vertical acrylamide gel using TEA buffer.A 4 % non denaturing gel is correct for most applications. 2.Run out DNA fragments.For fragments greater than 500 bprun the xylene cyanol dye to the b ...

I have read some books but cannot find the answer.I think NaAc may increase the ionic strength to stabilize DNA duplexes.Is it right? Thanks. -freshman- ----------------------------------------------- ...

TRANSFORMATION (ONE-STEP PEG METHOD) 1.Dilute 1:100 a fresh O/N culture of bacteria into prewarmed LB broth and incubate cells with shaking (225 rpm)to an OD600 of 0.3-0.4. 2.Add an equal volume of ic ...

Deproteination using phenol/chloroform (Maniatis et al.1982) 'Phenol' as used is Analar grade.Phenol should be melted at 65℃ 8-hydroxyquinoline added to a final concentration of 0.1%and equilibrated t ...

Use from 0.01 - 0.1 gram plant material. Grind the plant material with liq.N2 in a mortar.We normally use some alumina to crush hard tissue. Transfer the ground tissue to a eppendorf tube. Add 1 ml ex ...

Preparation of linear polyacrylamide used as carrier in ethanol precipitation of nucleic acids Linear polyacrylamide can be used as an efficient neutral carrier for precipitating nucleic acids with et ...

1、低温下长时间的连接效率比室温下短时间连接的好，平端连接需要过夜反应。 2、在体系中加一点切载体的酶，只要连接后原来的酶切位点消失。这样可避免载体自连，应该可以大大提高平端连接的效率。 足够多的载体和插入片段是最重要的 。要看片段具体情况而言，有时载体和片段浓度过高，容易产生线性化产物，不利于环化的。插入的DNA的摩尔数是载体摩尔数的5-10倍，这个很关键。载体通常50ng就够了，若载体在10k ...

Cepko/Tabin Lab Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/R1.html Protocol R.1 Random Prime Labeling of DNA Solutions Buffer A 1.25 M Tris 8.0 625 ml 2M Tris 8.0 2 125 ml 1M ...

hi thereI have been trying to clone 3.4 kb insert into pcDNA3.1- for months without any success.The insert is subcloned in TOPO from where I cut it out by KpnI and NotI and purify it by gelelectrophor ...

1.Crash Individual sand flies in 0.5 ml lysis buffer (50 mM NaCl 10 mM EDTA50 mM Tris-HCl pH 8).2.Incubate The sand fly homogenates with 100 ng/ml Rnase at 37 ℃ for 30 minutes. 3.Incubate The cell lys ...

Cepko/Tabin Lab Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/S6.html Superfrost plus slides and standard slides can be silinized but I have found that superfrost slides work be ...

A protocol / method / schedule /procedure for extraction / isolation of both DNA and RNA from the same material typically plant leaf / leaves (See also 1)Take one medium sized leaf or half a large lea ...

1.Add an equal volume (equal to sample volume)of P/C to sample. 2.Mix (shakedon't vortex). 3.Take aqueous (upper)layer.(If dirty samplerepeat Ph/Chl step until interface is fairly clean). 4.Add equal ...