分子生物学第九章-分子生物学技术-techniques of molecular biology
相关主题
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
(2) RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility.
(3) RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility only correlates with the molecular weight
sequences.(剪刀)
RE used in molecular biology typically recognize (识别) short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences.
1. Gel Electrophoresis （凝胶电泳）
Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties
DNA gel mobility (electrophoretic mobility ， DNA在胶上的迁移率)
1.DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶 支持物) toward the positive pole (正电极)
2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules.
large moderate small Run gel(跑胶)
3.The mobility of circular DNA molecules is affected by their topological structures.
The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋)> linear (线性) > nicked or relaxed (缺刻或松散)
2. Restriction endonucleases (限制 性内切酶) cleave DNA molecules at particular sites
Why use endonucleases? To make large DNA molecules break into manageable fragments
(2) but can separate DNA over a narrow size range (up to a few hundred bp, 几百bp).
Agarose (琼脂糖):
(1) a much less resolving
4 kb
power than
3 kb
polyacrylamide
Gel matrix (胶支持物)
Gel matrix (胶支持物) is a jello(果冻状）like porous material that supports and allows macromolecules to move through.
Polyacrylamide (聚丙烯酰胺)
Why use restriction endonucleases? cleave DNA molecules at particular sites
Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific
Agarose (琼脂糖)
Polyacrylamide (聚丙烯酰 胺):
(1) has high resolving capability(分辨力高), and can resolve DNA/RNA that differ from each other as little as a single base pair.
DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化 乙锭)
DNA marker
4 kb 3 kb 2 kb 1 kb
0.5 kb
DNA is separated by gel electrophoresis
2 kb
1 kb
0.5 kb
(2) but can separate DNA
molecules of up to
tens of kb
Electrophoresis is also used to separate RNAs
(1) RNA have a uniform negative charge as DNA does.
Pulsed-field gel electrophoresis (脉冲电泳)
百度文库12
pulsed-field gel electrophoresis (PFGE)
Switching between two orientations: the larger the DNA is, the longer it takes to reorient
(3) RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility only correlates with the molecular weight
sequences.(剪刀)
RE used in molecular biology typically recognize (识别) short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences.
1. Gel Electrophoresis （凝胶电泳）
Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties
DNA gel mobility (electrophoretic mobility ， DNA在胶上的迁移率)
1.DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶 支持物) toward the positive pole (正电极)
2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules.
large moderate small Run gel(跑胶)
3.The mobility of circular DNA molecules is affected by their topological structures.
The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋)> linear (线性) > nicked or relaxed (缺刻或松散)
2. Restriction endonucleases (限制 性内切酶) cleave DNA molecules at particular sites
Why use endonucleases? To make large DNA molecules break into manageable fragments
(2) but can separate DNA over a narrow size range (up to a few hundred bp, 几百bp).
Agarose (琼脂糖):
(1) a much less resolving
4 kb
power than
3 kb
polyacrylamide
Gel matrix (胶支持物)
Gel matrix (胶支持物) is a jello(果冻状）like porous material that supports and allows macromolecules to move through.
Polyacrylamide (聚丙烯酰胺)
Why use restriction endonucleases? cleave DNA molecules at particular sites
Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific
Agarose (琼脂糖)
Polyacrylamide (聚丙烯酰 胺):
(1) has high resolving capability(分辨力高), and can resolve DNA/RNA that differ from each other as little as a single base pair.
DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化 乙锭)
DNA marker
4 kb 3 kb 2 kb 1 kb
0.5 kb
DNA is separated by gel electrophoresis
2 kb
1 kb
0.5 kb
(2) but can separate DNA
molecules of up to
tens of kb
Electrophoresis is also used to separate RNAs
(1) RNA have a uniform negative charge as DNA does.
Pulsed-field gel electrophoresis (脉冲电泳)
百度文库12
pulsed-field gel electrophoresis (PFGE)
Switching between two orientations: the larger the DNA is, the longer it takes to reorient