y*d
2 楼
Taq酶的特征之一是会在PCR之后在3'端加上polyA尾,这方便了作TA克隆,但是我现在
的实验不希望有这个polyA尾,是否什么办法把它去掉,变成和pfu类似的平末端?谢谢!
补充:实验必须用Taq酶,不能换成pfu或Phusion之类的酶。
的实验不希望有这个polyA尾,是否什么办法把它去掉,变成和pfu类似的平末端?谢谢!
补充:实验必须用Taq酶,不能换成pfu或Phusion之类的酶。
y*d
5 楼
是的,不想要polyA,只是到引物的末端就可以了。
能否详细说说pfu处理的条件?是否需要先把Taq酶除去?
有相关的文献或资料吗?感谢!
能否详细说说pfu处理的条件?是否需要先把Taq酶除去?
有相关的文献或资料吗?感谢!
g*5
8 楼
only one A would be added on to PCR product.
and CloneJET PCR Cloning Kit have DNA Blunt enzyme.
and CloneJET PCR Cloning Kit have DNA Blunt enzyme.
y*d
11 楼
感谢大家的帮助和讨论,再多问两句:
1. 加入pfu后的反应温度应该是多少?室温,37度,还是72度比较好?
2. 大概需要反应多长时间?是否有办法确定反应完毕?
对这个领域不太熟,见笑了!
1. 加入pfu后的反应温度应该是多少?室温,37度,还是72度比较好?
2. 大概需要反应多长时间?是否有办法确定反应完毕?
对这个领域不太熟,见笑了!
M*n
12 楼
use T4 DNA polymerase without dNTP,
it will trim the overhang,
double-check manufacturter's manual, because I may have remembered it wrong.
it will trim the overhang,
double-check manufacturter's manual, because I may have remembered it wrong.
y*d
13 楼
谢谢!查了一下说明,发现这些酶都是需要在dNTP存在来切除overhang的。如果没有
dNTP的情况下容易造成3'端的凹陷。
dNTP的情况下容易造成3'端的凹陷。
l*1
14 楼
RE LZ:
pls try
Cassette-ligation downstream 3'RACE PCR
or Tail-PCR
Protocol:
看图说话
>
Amplification of FSTs by 3’-RACE
Total RNA was isolated using hot-phenol extraction
[27], from 20 ml cultures in 50 ml Falcon tubes on a rotating
wheel. Reverse transcription used the SuperScript
III First-Strand kit from Invitrogen in a DNA Engine
PCR machine (MJ research). Primer QT was mixed with
5 μg RNA, incubated at 65 °C for 5 min then cooled to
55 °C over 100 sec, after which the annealed mixture
was kept at 0 °C. The RT-mix was added and incubation
proceeded at 25 °C for 5 min, then 50 °C for 50 min.
After inactivation at 85 °C (5 min) and cooling to 0 °C,
RNAseH was added and left to degrade template RNA
for 20 min at 37 °C. The resulting cDNA was diluted
20-fold in TE buffer and kept at 4 °C.
Refeence:
Meslet-Cladière L, Vallon O. (2012)
A new method to identify flanking sequence tags in chlamydomonas using 3'-
RACE.
Plant Methods. 8: 21.
web link:
http://www.ncbi.nlm.nih.gov/pubmed/22735168
【在 y****d 的大作中提到】
: 谢谢!查了一下说明,发现这些酶都是需要在dNTP存在来切除overhang的。如果没有
: dNTP的情况下容易造成3'端的凹陷。
pls try
Cassette-ligation downstream 3'RACE PCR
or Tail-PCR
Protocol:
看图说话
>
Amplification of FSTs by 3’-RACE
Total RNA was isolated using hot-phenol extraction
[27], from 20 ml cultures in 50 ml Falcon tubes on a rotating
wheel. Reverse transcription used the SuperScript
III First-Strand kit from Invitrogen in a DNA Engine
PCR machine (MJ research). Primer QT was mixed with
5 μg RNA, incubated at 65 °C for 5 min then cooled to
55 °C over 100 sec, after which the annealed mixture
was kept at 0 °C. The RT-mix was added and incubation
proceeded at 25 °C for 5 min, then 50 °C for 50 min.
After inactivation at 85 °C (5 min) and cooling to 0 °C,
RNAseH was added and left to degrade template RNA
for 20 min at 37 °C. The resulting cDNA was diluted
20-fold in TE buffer and kept at 4 °C.
Refeence:
Meslet-Cladière L, Vallon O. (2012)
A new method to identify flanking sequence tags in chlamydomonas using 3'-
RACE.
Plant Methods. 8: 21.
web link:
http://www.ncbi.nlm.nih.gov/pubmed/22735168
【在 y****d 的大作中提到】
: 谢谢!查了一下说明,发现这些酶都是需要在dNTP存在来切除overhang的。如果没有
: dNTP的情况下容易造成3'端的凹陷。
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