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Restriction Endonuclease Digestion of DNA
限制性内切酶 DNA 消化

Outline:Restriction endonuclease digestion is a commonly used method for the cutting up of DNA (plasmid, PCR product or genomic DNA) into fragments for purposes including cloning or for restriction enzyme mapping. The choice of restriction enzyme used typically depends upon factors including the sequence(s) that you wish to cut, the target vector that you are using for cloning and, in the case of more than one enzyme being used, and the availability of compatible buffers. Different enzymes can require different buffers and need different incubation and inactivation temperatures, so reading the information leaflet accompanying an enzyme is essential
概述:限制性核酸内切酶消化是将 DNA(质粒、PCR 产物或基因组 DNA)切割成片段的常用方法,用于克隆或限制性内切酶作图等目的。所用限制性内切酶的选择通常取决于多种因素,包括您希望切割的序列、用于克隆的目标载体,如果使用多种酶,以及兼容缓冲液的可用性。不同的酶可能需要不同的缓冲液,并且需要不同的孵育和灭活温度,因此阅读酶随附的信息手册是必不可少的
.

Materials, reagents and equipment
材料、试剂和设备

Plasmid DNA to be Restricted
限制DNA

Restriction endonuclease
限制性核酸内切酶

10x Buffer
10x 缓冲液

Bovine Serum Albumin (BSA) (if needed, or if not in the 10x Buffer)
牛血清白蛋白 (BSA)(如果需要,或者如果不在 10x 缓冲液中)

Ice bucket, (Please collect the ice form the ice machine)
冰桶,(请从制冰机收集冰块)

Dry Incubator or Water bath at a suitable temperature
在合适的温度下干燥培养箱或水浴

Eppendorf tubes 1.5 ml (sterilised)
Eppendorf 管 1.5 ml(灭菌)

Pipette tips (sterilised)
移液器吸头(消毒)

Protocol:
协议:

A typical 20 µl digestion is shown here. Keep all components chilled on ice until the incubation / inactivation steps.
这里显示了典型的 20 μl 消化。将所有组分在冰上冷却,直到孵育/灭活步骤。

Thaw 10x restriction enzyme buffer and Bovine Serum Albumin (BSA) (if required).
解冻 10x 限制性内切酶缓冲液和牛血清白蛋白 (BSA)(如果需要)。

In a sterile 1.5 ml microcentrifuge tube add:
在无菌的 1.5 ml 微量离心管中加入:

Plasmid DNA (~0.5 µg undigested)
质粒 DNA(~0.5 μg 未消化)

1 µl
1微升

Buffer H1 (10x)
缓冲液 H110x)

2 µl
2 微升

BSA2 (20x) (optional)
BSA2 (20x)(可选)

1 µl
1 微升

Restriction enzyme (10 – 20 Unit / µl)3
限制性内切酶 (10 – 20 单位/μl)3

1 µl
1 微升

ddH2O to make up volume up to 20 µl
ddH2O 以补充高达 20 μl 的体积

X µl
X 微升

Total

20 µl
20 微升

Incubate 1 hour at 37°C4
在 37°C 下孵育 1 小时4
.

Place at 65°C for 20 minutes to inactivate the enzyme (optional)4
在 65°C 下放置 20 分钟以灭活酶(可选)4

Notes:
笔记:

1The choice of buffer depends upon the enzyme used. Pick a buffer that allows your enzyme to have a high activity (usually the lids of the enzyme and buffer are colour co-ordinated). Avoid buffers that have a ‘high star activity’, which means non-specific.
1缓冲液的选择取决于所使用的酶。选择一种能使酶具有高活性的缓冲液(通常酶和缓冲液的盖子颜色协调)。避免使用具有 “high star activity” 的缓冲液,这意味着非特异性。

2Some companies include BSA in their standard buffers, whilst some have it in a separate tube to add here. Read the protocol!
2一些公司在其标准缓冲液中加入了 BSA,而有些公司则将其放在单独的试管中,以便在此处添加。阅读协议!

3One unit is the amount of enzyme needed to cut 1µg lambda DNA in 1 hour at 37°C. Add the enzyme last. Never allow to total amount of enzyme in a reaction to exceed 10% of the final volume as this causes star activity
31 单位是在 37°C 下 1 小时内切割 1 μg λ DNA 所需的酶量。 最后添加酶 切勿使反应中酶的总量超过最终体积的 10%,因为这会导致星号活性
.

4These temperatures are very much enzyme dependent so read the product literature.
4这些温度在很大程度上依赖于酶,因此请阅读产品文献。

References:
引用:

https://www.promega.co.uk/resources/product-guides-and-selectors/restriction-enzyme-resource/restriction-enzyme-general-information/

https://www.promega.co.uk/~/media/files/resources/protocols/technical%20manuals/101/restriction%20enzymes%20protocol.pdf

Agarose gel electrophoresis of DNA
DNA 的琼脂糖凝胶电泳

Outline: Agarose gel electrophoresis is used for the resolution, visualisation and also for quantitation of DNA fragments. The DNA that is run can be a single species (i.e. PCR product) or a complex population (i.e. digested genomic DNA). Agarose gel electrophoresis can be used for diagnostic purposes (i.e. checking the size of an insert in a plasmid), for preparative purposes (i.e. for gel extraction), or just to estimate the amount of DNA present in a single band.
概述:琼脂糖凝胶电泳用于 DNA 片段的分辨率、可视化 定量。运行的 DNA 可以是单个物种(即 PCR 产物)或复杂群体(即消化的基因组 DNA)。琼脂糖凝胶电泳可用于诊断目的(即检查质粒中插入片段的大小)、制备目的(即用于凝胶提取)或仅用于估计单个条带中存在的 DNA 量。

We use Midori Green Nucleic Acid Staining Solution that is a new and safe alternative to traditional ethidium bromide (EtBr) stain for detecting dsDNA, ssDNA and RNA in agarose gels. Nearly identical to EtBr in performance and use, Midori Green is much less harmful to living organisms.
我们使用 Midori Green 核酸染色液,这是一种新型且安全的替代传统溴化乙锭 (EtBr) 染色剂,用于检测琼脂糖凝胶中的 dsDNA、ssDNA 和 RNA。Midori Green 在性能和使用上与 EtBr 几乎相同,对生物体的危害要小得多。

Materials:
材料:

50x TAE (40 mM tris-base, 20 mM acetate, 1 mM EDTA; pH between 8.2-8.4).
50x TAE(40 mM tris-碱、20 mM 乙酸盐、1 mM EDTA;pH 值在 8.2-8.4 之间)。

6x DNA loading buffer (30% glycerol with a dash of bromophenol blue to colour)
6x DNA 上样缓冲液(30% 甘油加少许溴酚蓝至颜色)

DNA ladder (also call DNA markers) Usually we use 1 Kb DNA Hyper ladder
DNA 分子量标准(也称为 DNA 标记物)通常我们使用 1 Kb DNA Hyper 分子量标准

Midori Green Nucleic Acid Staining Solution
Midori Green 核酸染色液

Power supply
电源

Gel tank, casting tray and comb
凝胶槽、灌铸盘和电泳梳

Masking tape
遮蔽胶带

Agarose
琼脂 糖

Microwave
微波

Transilluminator / Gel Documentation System
透射仪/凝胶成像系统

Protocol for 100 ml of 1% (w/v) agarose:
100 ml 1% (w/v) 琼脂糖的方案:

Prepare sufficient 1x TAE for making the gel and filling the gel tank. Dilute the 50x TAE stock solution down with distilled water to 1x.
准备足够的 1x TAE 来制备凝胶和填充凝胶槽。用蒸馏水将 50x TAE 储备液稀释至 1x。

Place 100 ml of 1x TAE and 1 g electrophoresis-grade agarose in a large microwaveable vessel and melt it in a microwave on a setting of around 40% until the agarose is completely dissolved. **** CAUTION – THIS IS VERY HOT ****
将 100 ml 1x TAE 和 1 g 电泳级琼脂糖置于一个大的可微波容器中,并在微波炉中以约 40% 的设置熔化,直到琼脂糖完全溶解。 注意 – 这很热 ****

Allow the molten agarose to cool until approximately 60°C (hand-hot) in a water-bath, or at room temperature shaking regularly (Optional). Whilst cooling, tape up the casting tray and add the comb.
让熔融琼脂糖在水浴中冷却至约 60°C(手热),或在室温下定期摇晃(可选)。冷却时,用胶带粘住铸盘并加入梳子。

When cooled, add 6 µl Midori Green dye (for 100 ml agarose), and mix by swirling (try not to introduce bubbles).
冷却后,加入 6 μl Midori Green 染料(用于 100 ml 琼脂糖),并通过旋转混合(尽量不要引入气泡)。

Pour the gel into the tray. If bubbles are present, burst them using the wide end of a P200 pipette tip. Allow the gel to solidify at room temperature.
将凝胶倒入托盘中。如果存在气泡,请使用 P200 移液器吸头的宽端将其爆裂。让凝胶在室温下凝固。

When cooled, place the gel in the gel tank and pour in 1x TAE until it is covered. Remove the comb (note: removing it when the gel is submerged helps prevent the wells being damaged).
冷却后,将凝胶放入凝胶槽中,倒入 1x TAE 直至被覆盖。取下电泳梳(注意:将电泳梳浸入水中时将其取出有助于防止损坏电泳槽)。

Prepare your samples. Only DNA bands >10 ng can be visualised. For PCR products this typically 5 µl of a PCR reaction is run, whilst 5 µl of a miniprep is also run. Add water to bring each sample up to 10 µl and then add 2 µl of 6x loading dye.
准备样品。只能观察到 >10 ng 的 DNA 条带。对于 PCR 产物,通常运行 5 μL 的 PCR 反应,同时运行 5 μL 的小量制备。加水使每个样品达到 10 μl,然后加入 2 μl 6x 上样染料。

In one well of the gel add 5 µl of DNA ladder.
在凝胶的一个孔中加入 5 μL DNA 分子量标准。

In the other wells load the samples that you wish to analyse.
在其他孔中加载您要分析的样品。

Put the lid on the tank, connect up the tank to the power supply and run at 100V for one hour. NB. DNA is negatively charged and runs towards the positive electrode. **** BE CAREFUL WITH MIXING WATER AND ELECTRICITY ***** Stop the gel early if you think that it has run far enough.
盖上水箱盖,水箱连接到电源,以 100V 电压运行一小时。铌。DNA 带负电并流向正极。 小心混合水和电 ***** 如果您认为凝胶已经运行得足够远,请尽早停止凝胶。

Turn the power supply off, and wearing gloves remove the gel from the tank.
关闭电源,戴上手套从电泳槽中取出凝胶。

Take the gel to the transilluminator and visualise the gel. **** UV light is mutagenic, always use a transilluminator cabinet and follow the instructions given ****
将凝胶带到透射仪上并观察凝胶。 紫外线具有致突变作用,请始终使用透射仪柜并按照给出的说明进行操作 ****

Sizes of bands in your samples can be estimated from the marker lanes. Because each marker band contains a known amount of DNA, comparison of the band brightness allows the quantity of DNA to be found.
样品中条带的大小可以从标记泳道中估计出来。由于每个标记条带都包含已知量的 DNA,因此比较条带亮度可以找到 DNA 的数量。

Notes: Choose the concentration of agarose in the gel depending on the size of the fragments you wish to resolve.
注意:根据要分离的片段大小选择凝胶中琼脂糖的浓度。

Agarose (%)
琼脂糖 (%)

Optimal band resolution
最佳频带分辨率

0.5

700 bp to 25 kbp
700 bp 至 25 kbp

0.8

500 bp to 15 kbp
500 bp 至 15 kbp

1.0

250 bp to 12 kbp
250 bp 至 12 kbp

1.2

150 bp to 6 kbp
150 bp 至 6 kbp

1.5

80 bp to 4 kbp
80 bp 至 4 kbp

Different types of agarose are available, suitable for different functions; for virtually all purposes a standard electrophoresis grade agarose will suffice
有不同类型的琼脂糖可供选择,适用于不同的功能;对于几乎所有目的,标准电泳级琼脂糖就足够了

References:
引用:

Sambrook, J. & Russell, D.W. (2001). Chapter 4: Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis. In: Molecular Cloning: A Manual. 3rd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. pp. 5.1-5.17.
Sambrook, J. & Russell, D.W. (2001)。第 4 章:DNA 凝胶电泳和脉冲场琼脂糖凝胶电泳。在:分子克隆:手册。第 3 版,冷泉港,纽约:冷泉港实验室出版社,第 5.1-5.17 页。