m****n
发帖数: 1066 | 1 1.PCR product is about 200bp. PCR product was run on an agarose gel, cut off
from the gel and purified using a Qia quick gel extraction kit from Qiagen
. The elution buffer doesn’t have EDTA.
2.PCR primers were used as sequencing primers.
3.During the first try, some part of the sequence was readable. The
remaining was mixed with NNNN.
4.During the second try, I redid the PCR and gel-purification. All of the
sequences were NNN. The same sequencing contractor was used.
I like to make some changes since I have been doing this for 2 weeks.
1.Change to another sequencing provider.
2.Change elution buffer to water.
3.Use nest primers as sequencing primers. Nest primers will be several bases
inward.
Anything else can I do?
Thanks! |
s******s
发帖数: 13035 | 2 直接测胶回收的PCR片段问题很多，我们这里facility基本上
就是告诉你别这么做。最好TA克隆了以后再测。
一定要测，我感觉要注意以下几点
1. UV照射时间尽量短。
2. Gel回收以后再PCR purification或者MinElute一遍，直接
gel extraction kit出来的东西非常脏。除非你QG/PE洗上十遍
八遍。
3. 测序引物可能缩进一点会好一些
off
Qiagen
【在 m****n 的大作中提到】
: 1.PCR product is about 200bp. PCR product was run on an agarose gel, cut off
: from the gel and purified using a Qia quick gel extraction kit from Qiagen
: . The elution buffer doesn’t have EDTA.
: 2.PCR primers were used as sequencing primers.
: 3.During the first try, some part of the sequence was readable. The
: remaining was mixed with NNNN.
: 4.During the second try, I redid the PCR and gel-purification. All of the
: sequences were NNN. The same sequencing contractor was used.
: I like to make some changes since I have been doing this for 2 weeks.
: 1.Change to another sequencing provider.
|
n******d
发帖数: 680 | 3 没有觉得阿，上周刚在楼下的facility测了两个PCR片段，一个700bp一个450bp，单引
物测序，结果都非常不错的阿。
我只用了Qiagen的kit做了一次胶回收纯化，然后用30 ul的dH2O洗脱，最后用乙醇浓缩
了一下下。因为我这里的测序的要求是，PCR产物浓度应该达到10ng/ul。
【在 s******s 的大作中提到】
: 直接测胶回收的PCR片段问题很多，我们这里facility基本上
: 就是告诉你别这么做。最好TA克隆了以后再测。
: 一定要测，我感觉要注意以下几点
: 1. UV照射时间尽量短。
: 2. Gel回收以后再PCR purification或者MinElute一遍，直接
: gel extraction kit出来的东西非常脏。除非你QG/PE洗上十遍
: 八遍。
: 3. 测序引物可能缩进一点会好一些
:
: off
|
k****l
发帖数: 279 | 4 Qiaquick doesn't give you very clean DNA, I also keep failing in it. If you
really don't want to clone it, my gel purification from GeneClean III kit
usually works
off
Qiagen
【在 m****n 的大作中提到】
: 1.PCR product is about 200bp. PCR product was run on an agarose gel, cut off
: from the gel and purified using a Qia quick gel extraction kit from Qiagen
: . The elution buffer doesn’t have EDTA.
: 2.PCR primers were used as sequencing primers.
: 3.During the first try, some part of the sequence was readable. The
: remaining was mixed with NNNN.
: 4.During the second try, I redid the PCR and gel-purification. All of the
: sequences were NNN. The same sequencing contractor was used.
: I like to make some changes since I have been doing this for 2 weeks.
: 1.Change to another sequencing provider.
|
d*******j
发帖数: 64 | 5 在你的一个引物5‘端加上M13序列，pcr后不要切胶回收，做个cleanup或者直接拿去测
序，测序引物用M13。
off
Qiagen
【在 m****n 的大作中提到】
: 1.PCR product is about 200bp. PCR product was run on an agarose gel, cut off
: from the gel and purified using a Qia quick gel extraction kit from Qiagen
: . The elution buffer doesn’t have EDTA.
: 2.PCR primers were used as sequencing primers.
: 3.During the first try, some part of the sequence was readable. The
: remaining was mixed with NNNN.
: 4.During the second try, I redid the PCR and gel-purification. All of the
: sequences were NNN. The same sequencing contractor was used.
: I like to make some changes since I have been doing this for 2 weeks.
: 1.Change to another sequencing provider.
|
s******s
发帖数: 13035 | 6 ft。你胶回收了以后，又用乙醇沉淀了一下，就是相当于第二次纯化了。所以我说那个
回收出来的太脏。
【在 n******d 的大作中提到】
: 没有觉得阿，上周刚在楼下的facility测了两个PCR片段，一个700bp一个450bp，单引
: 物测序，结果都非常不错的阿。
: 我只用了Qiagen的kit做了一次胶回收纯化，然后用30 ul的dH2O洗脱，最后用乙醇浓缩
: 了一下下。因为我这里的测序的要求是，PCR产物浓度应该达到10ng/ul。
|
m****n
发帖数: 1066 | 7 乙醇浓缩后你用的水溶解？
【在 n******d 的大作中提到】
: 没有觉得阿，上周刚在楼下的facility测了两个PCR片段，一个700bp一个450bp，单引
: 物测序，结果都非常不错的阿。
: 我只用了Qiagen的kit做了一次胶回收纯化，然后用30 ul的dH2O洗脱，最后用乙醇浓缩
: 了一下下。因为我这里的测序的要求是，PCR产物浓度应该达到10ng/ul。
|
m****n
发帖数: 1066 | |
Z******5
发帖数: 435 | 9 一般测序貌似前50bp信号都不好。你一共200bp，有1/4测不好。当然也要考虑一下前面
50bp是否是你的关键序列。 双向测序倒是可以解决。 |
Z******5
发帖数: 435 | 10 测序前最好用PCR产物纯化试剂盒纯化。至于用水洗还是用kit的洗脱液，都无关紧要。 |
