Screw Tissue Press
The Screw Tissue Press is used as an initial tissue dispersion step before lysing fresh tissue1 with a mechanical homogenizer (ultrasonicator, beadbeater, rotor-stator, etc) or the preparation of single cells. Chopping tissue into pieces with scissors or scalpel is eliminated, and much smaller tissue fragments are produced. Unlike other tissue dispersion/blending devices, no buffer or media is added during its operation. The dispersed tissue exits out of the end of the Screw Tissue Press has a consistency much like liver paté or toothpaste.
The Screw Press also is used to prepare viable single cells, either directly2 or as a first step via enzyme digestion of the extracellular connective matrix. The benefits of an initial Screw Press disaggregation of tissue is reduced incubation times with the digestive enzymes and increased diffusion of critical nutrients and gases into tissue cells during the incubation period. The net result is higher yields of viable cells. [ For an excellent general review see Methods of Enzymatic Tissue Disaggregation ].
1. Tissue processed in this press must be "soft" (i.e., relatively non-fibrous and have high water content). Acceptable examples are muscle, brain, liver, plant fruits, some plant roots and tubers.
2. Example:>A rapid method for the preparation of single‐cell suspensions from solid tissues, NP Singh - Cytometry 31: 229–232, 1998
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Operating Instructions:
1) Acceptible tissue is soft, relatively non-fibrous and has high water content. Examples are muscle, brain, liver, plant fruits, some plant roots and tubers. If the tissue is partially fibrous, fatty or covered with a tough membrane, first manually trim off the tough or fatty parts. Fibrous tissue will interfere with the passage of the soft tissue through the press and lower the yield of dispersed cell material. Skin, hide, connective tissue, plant stems, wood and dried tissues cannot be processed.
2) Partially retract the piston cylinder of the Screw Tissue Press to make room for the tissue. Unscrew the 'cap' with its array of holes from the bottom of the Screw Tissue Press and place the tissue sample into the cavity of the press.
Generally, the array of 2 mm diameter holes in the press cap is used. If your sample is soft, non-fibrous material with high water content, you can also use the included 500 micron stainless steel wire sieve. Carefully center the screen on the end of the press cylinder after adding the sample tissue and screw on the press cap.
3) Manually turn the piston to extrude the tissue. The dispersed tissue will exit the press having a paste- or granular-like texture. Depending on the amount of connective or fibrous tissue present in the sample, the yield of dispersed tissue will vary from 20-95%.
4) Extruded tissue material can be removed from the end of the Screw Tissue Press with a scalpel or single edge razor blade.
Color | Silver |
Hardware | 32GB |
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DNA连接酶主要是连接DNA片段之间的磷酸二酯键最初从原核生物(大肠杆菌)分离得到的.现在生物基因工程主要是从T4噬菌体中分离得到的,
酶对所作用的底物有严格的选择性。一种酶仅能作用于一种物质,或一类分子结构相似的物质,促其进行一定的化学反应,产生一定的反应产物,这种选择性作用称为酶的专一性。
连接酶通常是包括“连接酶”这个字,就如DNA连接酶是将脱氧核糖核酸(DNA)片段连接。其他普遍的名称包括“合成酶”,因为这些酶是用作合成新的分子,或当它们是将二氧化碳加入一个分子时则称为“羧化酶”。
菌体构建时,目的基因连接在T载体上,测序也正确,但是将目的基因还有载体分别双酶切后总是连接不上,将连接后产物跑核酸胶,什么也没有,不知道是什么原因?我的目的基因浓度为9ng/ul,载体回收后的浓度为6ng/ul,是因为浓度太低的原因吗?还有就是连接有目的基因的T载体双酶切后出现了三条带,这个又是什么原因呢?希望得到解答
大家有用过Invitrogen的T4连接酶吗?说明书上是23-26度连接,一般的连接酶不都是16度吗?应该用多少度呢?另外,说明书上还说连接后为了达到更好的转化效率,应将连接反应液至少稀释5倍再转化,是这样吗?谢谢大家帮忙啊
连接酶有T4噬菌体DNA连接酶、T4噬菌体RNA连接酶、大肠埃希菌DNA连接酶等。DNA连接酶可连接平端,也连接粘端。反应需有Mg2+和ATP存在,pH7.5-7.6。最适温度37℃,30℃以下活性明显下降,但考虑到被连接DNa 的稳定性和粘性末端的退火温度,一般平端连接用20-25℃,粘端连接用12℃左右。
聚合酶有DNA聚合酶(以DNA为模板合成DNA大肠埃希菌DNA聚合酶Ⅰ,大肠埃希菌DNA聚合酶Ⅰ大片段(Klenow大片段),T4或T7噬菌体DNA聚合酶等);RNA聚合酶(以DNA为模板合成RNA,T7或T3噬菌体RNA聚合酶);逆转录酶(以RNA为模板合成DNA,除RNA病毒中发现外,发现大肠埃希菌DNA聚合酶Ⅰ和Taq DNA聚合酶都有逆转录活性)。
大肠杆菌DNA聚合酶Ⅰ具有5′→3′聚合酶活性和5′→3′,3′→5′外切酶活性。Klenow片段是DNA聚合酶Ⅰ被枯草杆菌酶作用产生的一个大片段,有5′→3′聚合酶和3′→5′外切酶活性,无5′→3′外切酶活性。可用于缺口翻译(Nick translation)法标记核酸,也可用于DNA序列测定,修补DNA链等。向左转|向右转
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