a******e
发帖数: 119 | 1 我在用Taq DNA ligase将两个富含GC的约70bp的单链DNA片段连接。 直接连，没连上。
又95度加热10分钟，cool down overnight，再连接，还是没连上。 请教各位有什么建
议么？
另外如果把单链变为双链后再连接，怎么由双链的DNA在获得单链。 Thanks |
s******y
发帖数: 28562 | 2 FT! FT! FT!
Of course you cannot ligate the single strand DNA like this way! Because
there is NOTHING to hold these two fragments together to allow the ligase to
act.
If you really want to ligate the single strand DNA, at least you should use
some kind of carrier system like BSA or other stuffs like a non-specific DNA
-binding protein.
If you choose to ligate from the double strand DNA, you can of course break
it down to single strand by incubating them on high temperature with
denaturing reagen
【在 a******e 的大作中提到】
: 我在用Taq DNA ligase将两个富含GC的约70bp的单链DNA片段连接。 直接连，没连上。
: 又95度加热10分钟，cool down overnight，再连接，还是没连上。 请教各位有什么建
: 议么？
: 另外如果把单链变为双链后再连接，怎么由双链的DNA在获得单链。 Thanks
|
a***e
发帖数: 1010 | 3 you need a bridge DNA.
check NEB
Taq DNA Ligase catalyzes the formation of a phosphodiester bond between
juxtaposed 5′ phosphate and 3′ hydroxyl termini of two adjacent
oligonucleotides which are hybridized to a complementary target DNA. The
ligation will occur only if the oligonucleotides are perfectly paired to the
complementary target DNA and have no gaps between them; therefore, a single
-base substitution can be detected. Taq DNA Ligase is active at elevated
temperatures (45°C-65°C) (1,2). |
a******e
发帖数: 119 | 4 sorry， 忘记说明了， 我用了complementary target DNA。 我用的反应条件是室温，
反应2个小时。
the
single
【在 a***e 的大作中提到】
: you need a bridge DNA.
: check NEB
: Taq DNA Ligase catalyzes the formation of a phosphodiester bond between
: juxtaposed 5′ phosphate and 3′ hydroxyl termini of two adjacent
: oligonucleotides which are hybridized to a complementary target DNA. The
: ligation will occur only if the oligonucleotides are perfectly paired to the
: complementary target DNA and have no gaps between them; therefore, a single
: -base substitution can be detected. Taq DNA Ligase is active at elevated
: temperatures (45°C-65°C) (1,2).
|
