w******e
发帖数: 1187 | 1 I suspected my dsDNA (100bp) pool (random sequence) dissociate and
form a messy mesh structure. When I run sample on a gel, many bands
show up above the expected molecular weight.
Anybody has exp with this problem? Thanks! |
s***e
发帖数: 911 | 2 这个不可能. 100 bp DNA已经非常稳定了. 100 bp DNA成环后也不会melt, 何况你的是
线性DNA.
【在 w******e 的大作中提到】
: I suspected my dsDNA (100bp) pool (random sequence) dissociate and
: form a messy mesh structure. When I run sample on a gel, many bands
: show up above the expected molecular weight.
: Anybody has exp with this problem? Thanks!
|
s***e
发帖数: 911 | 3 oh. 你这个是100 bp DNA 环吗?
【在 w******e 的大作中提到】
: I suspected my dsDNA (100bp) pool (random sequence) dissociate and
: form a messy mesh structure. When I run sample on a gel, many bands
: show up above the expected molecular weight.
: Anybody has exp with this problem? Thanks!
|
h****6
发帖数: 229 | 4 If it is high AT DNA, DNA may melt during gel running. If DNA melts, I
doubt it will re-form dsDNA under the same condition. ssDNA migrate slower. |
s***e
发帖数: 911 | 5 如果是环, 100 bp的DNA在这么高的曲率下, 可能会有局部melting (one or a few bp)
来环解弯曲弹性能.在跑胶的温度下, 这样的local melting sites可能会有数个. 不同
的个数会导致DNA不同的构型, gel shift速度会不一样.
【在 s***e 的大作中提到】
: oh. 你这个是100 bp DNA 环吗?
|
w******e
发帖数: 1187 | 6 thx for the info. no it's not circular DNA. it's random pool, so if the
ds structure ever dissociates, there is virtually no chance for
re-annealing thou. I just don't know whether it's possible for the ds
structure to dissociate under normal experimental condition (like gel
running, or salt solution, etc)
bp)
【在 s***e 的大作中提到】
: 如果是环, 100 bp的DNA在这么高的曲率下, 可能会有局部melting (one or a few bp)
: 来环解弯曲弹性能.在跑胶的温度下, 这样的local melting sites可能会有数个. 不同
: 的个数会导致DNA不同的构型, gel shift速度会不一样.
|
s***e
发帖数: 911 | 7 不太可能. 单分子unzip DNA hairpin的测量里, DNA一般比100 bp还短. normal
experimental condition下需要> 10pN才能把双链分开.
【在 w******e 的大作中提到】
: thx for the info. no it's not circular DNA. it's random pool, so if the
: ds structure ever dissociates, there is virtually no chance for
: re-annealing thou. I just don't know whether it's possible for the ds
: structure to dissociate under normal experimental condition (like gel
: running, or salt solution, etc)
:
: bp)
|
a***e
发帖数: 1010 | 8 make fresh buffer and run the gel again.
make sure the loading buffer is correct.
generally, ssDNA runs faster than dsDNA with the same length. So should run
below, not above, your expected band. |
w******e
发帖数: 1187 | 9 thx again. my situation is: I gel purified some 100bp pool a while ago.
the pool looked great on the gel for extraction (sharp 100bp band).
I didn't bother to run gel again after the extraction (stupid of me)
and just spec them and threw them in the freezer. I noticed a high
salt peak at 230 but felt I don't have to care for now.
today (~2 months later) I took it out, mix w/ normal loading buffer,
and ran it on a native TBE-PAGE gel as mass standard(I always use this
type of gel for short dsDNA
【在 s***e 的大作中提到】
: 不太可能. 单分子unzip DNA hairpin的测量里, DNA一般比100 bp还短. normal
: experimental condition下需要> 10pN才能把双链分开.
|
s***e
发帖数: 911 | 10 我不知道DNA在power form有多稳定. 短DNA出现smear band我见过一次. 不过那是因为
这个DNA里有许多tandem sequence. 所以错位一个周期, 能量不是很大.
【在 w******e 的大作中提到】
: thx again. my situation is: I gel purified some 100bp pool a while ago.
: the pool looked great on the gel for extraction (sharp 100bp band).
: I didn't bother to run gel again after the extraction (stupid of me)
: and just spec them and threw them in the freezer. I noticed a high
: salt peak at 230 but felt I don't have to care for now.
: today (~2 months later) I took it out, mix w/ normal loading buffer,
: and ran it on a native TBE-PAGE gel as mass standard(I always use this
: type of gel for short dsDNA
|
|
|
w******e
发帖数: 1187 | 11 thx anyway. I'll report to the board if I found anything interesting:)
【在 s***e 的大作中提到】
: 我不知道DNA在power form有多稳定. 短DNA出现smear band我见过一次. 不过那是因为
: 这个DNA里有许多tandem sequence. 所以错位一个周期, 能量不是很大.
|
a****o
发帖数: 1786 | 12 I did not read all posts. I guess LZ is doing SELEX. What he/she refered is
the bulge structure formed by many different molecules share two constant
regions at both ends, but with different sequences in the middle.
The simplest way to solve this problem is to dilute your sample to 4 fold
after PCR, add do ONE more cycle of PCR. |
w******e
发帖数: 1187 | 13 thx for the input. PCR is a great idea. so did you see this kind of
dissociation happening in your exp? what are the factors that affect
the process? storage buffer? time? it's surprising to me as all the
newly prepared dsDNA pool have no such problem on gel.
is
【在 a****o 的大作中提到】
: I did not read all posts. I guess LZ is doing SELEX. What he/she refered is
: the bulge structure formed by many different molecules share two constant
: regions at both ends, but with different sequences in the middle.
: The simplest way to solve this problem is to dilute your sample to 4 fold
: after PCR, add do ONE more cycle of PCR.
|
a****o
发帖数: 1786 | 14 Yes, if you overdid PCR, you will see this phenomenon. Doing PCR for a
fewer cycles may cure the problem. I don't think it is dissociation. When
primer/dNTP/active Taq goes down, annealed DNA-Primer can not be
formed/extended. DNA molecules with different sequence in middle form
duplex. Since they have identical sequences at both ends due to constant
regions, but different sequences in the middle, they can not form perfect
duplex, rather they form partial duplex at both ends, while loop-out
sing
【在 w******e 的大作中提到】
: thx for the input. PCR is a great idea. so did you see this kind of
: dissociation happening in your exp? what are the factors that affect
: the process? storage buffer? time? it's surprising to me as all the
: newly prepared dsDNA pool have no such problem on gel.
:
: is
|
w******e
发帖数: 1187 | 15 thx a lot for the info. In fact, the product was after PCR optimization
and ran fine on the gel before band cutting and gel extraction, so I
don't think PCR bias is the reason. However, I do agree doing one more
round of PCR is the way to go. If that doesn't get s**t together, there
must be something really wrong with my hands...
【在 a****o 的大作中提到】
: Yes, if you overdid PCR, you will see this phenomenon. Doing PCR for a
: fewer cycles may cure the problem. I don't think it is dissociation. When
: primer/dNTP/active Taq goes down, annealed DNA-Primer can not be
: formed/extended. DNA molecules with different sequence in middle form
: duplex. Since they have identical sequences at both ends due to constant
: regions, but different sequences in the middle, they can not form perfect
: duplex, rather they form partial duplex at both ends, while loop-out
: sing
|
