j******i
发帖数: 939 | 1 最近实验室里很多人克隆时遇到了这个诡异的事情。
1. PCR产物跑胶。大小正确。
2. 切胶回收提纯之后，第二天再跑胶确认，DNA片段居然变小了（不是smear)。
这个怎么解释呢？
1. 细菌污染，DNA降解？那应该得到很多很小的片段才是，而现在确是一条很强的条带
，只是变小了而已。
2. 变成单链DNA了? 倒是解释的通，但是怎么变呢？
各位有何高见呢？ |
m*********D
发帖数: 1727 | 2 If you use water to dissolve the DNA, you might get single-stranded DNA.
To form DS-DNA, certain salt concentration is necessary. I generally mix the
DNA with TE buffer (1:9), and then load on gel with loading buffer. It will
get back to normal size. |
l********e
发帖数: 415 | 3 我也遇到过。当时也猜测是这个原因，用TE重复一次果然就没事了。
the
will
【在 m*********D 的大作中提到】
: If you use water to dissolve the DNA, you might get single-stranded DNA.
: To form DS-DNA, certain salt concentration is necessary. I generally mix the
: DNA with TE buffer (1:9), and then load on gel with loading buffer. It will
: get back to normal size.
|
w*****r
发帖数: 2061 | 4 想知道大小变了多少？DNA多大？
【在 j******i 的大作中提到】
: 最近实验室里很多人克隆时遇到了这个诡异的事情。
: 1. PCR产物跑胶。大小正确。
: 2. 切胶回收提纯之后，第二天再跑胶确认，DNA片段居然变小了（不是smear)。
: 这个怎么解释呢？
: 1. 细菌污染，DNA降解？那应该得到很多很小的片段才是，而现在确是一条很强的条带
: ，只是变小了而已。
: 2. 变成单链DNA了? 倒是解释的通，但是怎么变呢？
: 各位有何高见呢？
|
j******i
发帖数: 939 | 5 果然ok了！多谢！
the
will
【在 m*********D 的大作中提到】
: If you use water to dissolve the DNA, you might get single-stranded DNA.
: To form DS-DNA, certain salt concentration is necessary. I generally mix the
: DNA with TE buffer (1:9), and then load on gel with loading buffer. It will
: get back to normal size.
|
s******s
发帖数: 13035 | 6 loading buffer本身应该就是10xTE加dye啊
【在 l********e 的大作中提到】
: 我也遇到过。当时也猜测是这个原因，用TE重复一次果然就没事了。
:
: the
: will
|
j******i
发帖数: 939 | 7 有的loading buffer可能没有盐。我换了一种loading buffer就好了。
【在 s******s 的大作中提到】
: loading buffer本身应该就是10xTE加dye啊
|
j******i
发帖数: 939 | 8 ssDNA是怎么形成的呢。切出来的胶也就是用50来度加热啊，好像不至于都变性了啊。
the
will
【在 m*********D 的大作中提到】
: If you use water to dissolve the DNA, you might get single-stranded DNA.
: To form DS-DNA, certain salt concentration is necessary. I generally mix the
: DNA with TE buffer (1:9), and then load on gel with loading buffer. It will
: get back to normal size.
|
m*********D
发帖数: 1727 | 9 (ZT)
Salt concentration
The phosphate groups are moderately strong acids, and thus they are always
ionized at physiological pHs. The two oxygens are equivalent, so they share
the single negative charge. These negative charges tend to blow the double
helix structure apart, which is perhaps why I haven't included them in the
forces that hold the helix together. Why is the helix stable?
Inside the cell, the monovalent salt concentration is about 0.15 M. The
major cation in the cell is K, while in the diagram I snow Na (no matter).
The concentration of positive ions is greater than average around the
negative phosphates, and this screens the repulsive force between the
phosphates. Since the screen is dependent on the salt, the melting
temperature should be dependent on the salt concentration.
One of the better formulas for melting temperature [Richly and Rhoads,
Nucleic Acids Res 17: 8543, 1989] uses the nearest neighbor thermodynamic
data, i.e. del H and del S, from the same study quoted at the start of this
section (c is the oligonucleotide concentration).
Tm = (del H / (del S + R * ln (c/4)))- 273 + 16.6 * log (salt)
Thus, for every 10 fold decrease in salt concentration, the Tm decreases
about 17 degrees C. This means that most DNA will denature at room
temperature (20 C) when the salt concentration falls below 10-5 to 10-6 M.
【在 j******i 的大作中提到】
: ssDNA是怎么形成的呢。切出来的胶也就是用50来度加热啊，好像不至于都变性了啊。
:
: the
: will
|
j******i
发帖数: 939 | 10 原来这个道理！谢谢啊 ；）
share
【在 m*********D 的大作中提到】
: (ZT)
: Salt concentration
: The phosphate groups are moderately strong acids, and thus they are always
: ionized at physiological pHs. The two oxygens are equivalent, so they share
: the single negative charge. These negative charges tend to blow the double
: helix structure apart, which is perhaps why I haven't included them in the
: forces that hold the helix together. Why is the helix stable?
: Inside the cell, the monovalent salt concentration is about 0.15 M. The
: major cation in the cell is K, while in the diagram I snow Na (no matter).
: The concentration of positive ions is greater than average around the
|
l******a
发帖数: 3339 | 11 赞！！！
the
will
【在 m*********D 的大作中提到】
: If you use water to dissolve the DNA, you might get single-stranded DNA.
: To form DS-DNA, certain salt concentration is necessary. I generally mix the
: DNA with TE buffer (1:9), and then load on gel with loading buffer. It will
: get back to normal size.
|
