x********u 发帖数: 430 | 1 I have to design a forward primer consisting of restriction sites (8 base),
operator region (35 base), ribosome biding site (6 base), spacer sequence (
about 20 base) and the priming sequence (21 base) of my target gene. The
total lenth would be 100 base for this forward primer and the reverse primer
is just 30 base. Does anyone know if this primer set would be okay for PCR
amplifying my target gene (around 720bp)?
Thanks a lot. |
s******y 发帖数: 28562 | 2 可以做,但是条件得摸索一下。
我做过70+ 20 的组合条件,感觉杂带比较多。不过把正确带切出来再放大一次就好了。
,
primer
PCR
【在 x********u 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have to design a forward primer consisting of restriction sites (8 base), : operator region (35 base), ribosome biding site (6 base), spacer sequence ( : about 20 base) and the priming sequence (21 base) of my target gene. The : total lenth would be 100 base for this forward primer and the reverse primer : is just 30 base. Does anyone know if this primer set would be okay for PCR : amplifying my target gene (around 720bp)? : Thanks a lot.
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x********u 发帖数: 430 | 3 Thanks a lot, Sunnyday LaoShi!
了。
【在 s******y 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 可以做,但是条件得摸索一下。 : 我做过70+ 20 的组合条件,感觉杂带比较多。不过把正确带切出来再放大一次就好了。 : : , : primer : PCR
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n***w 发帖数: 2405 | 4 I never tried that long. I only tried 45 as the max.
You can also do overlap PCR so you can use short primer at every time. |
f******e 发帖数: 53 | 5 We found it is hard to get clear and single band using longer primer.
Now we normally design two overlapped PCR primer which each one is less than
50 bp in length.It works very well in my lab.
,
primer
PCR
【在 x********u 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have to design a forward primer consisting of restriction sites (8 base), : operator region (35 base), ribosome biding site (6 base), spacer sequence ( : about 20 base) and the priming sequence (21 base) of my target gene. The : total lenth would be 100 base for this forward primer and the reverse primer : is just 30 base. Does anyone know if this primer set would be okay for PCR : amplifying my target gene (around 720bp)? : Thanks a lot.
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C*******e 发帖数: 4348 | 6 理论上说应该没有问题
不过可能要摸条件
我用70-80,还有>100的primer做过regular PCR
不过我用的primer
1.Fwd和Rev差不多等长,Tm接近,
而且priming sequence (for target gene)部分的Tm也接近
2.我用的priming sequence比较长,24-28nts
我觉得你做的时候可能
1.不仅reaction的条件要摸
2.PCR cycle的设定要摸(除了gradient,还有touch down,touch up,3 steps, 2
steps之类的可以试)
3.有可能template也要试,我遇到过用整个plasmid不行,
把target gene的fragment切下来胶纯化以后做模板就没有问题
4.如果一切不顺利,建议reverse primer也设计个长点的
哦对了,还有一点,长的那个primer,最好要订纯化过的
不用HPLC纯化的也要PAGE纯化的
或者订了std desalted的回来自己用PAGE胶纯化一下
越长的primer越有可能5'段少一些(对于>100 nts的primer可能少的是一截)
甚至靠近5'段有些deletion,mutation也是可能的
如果不纯化,后面你挑positive clones去测序的时候就该郁闷了 |
h***e 发帖数: 146 | 7 我们经常做red重组 引物一般都70-100bp
没问题
如果是以质粒为模板 基本没杂带
要是一复杂的genomic DNA为模板 就不太好弄 |
b******n 发帖数: 4225 | 8 可以分两到三次PCR,毕竟长引物价格贵而且容易合成错误
第一次用RBS+SPACE+Priming sequencing作为forward
得到的产物纯化一下作为第二次PCR的模板
第二次用restriction+operator+RBS+Space作为forward
,
primer
PCR
【在 x********u 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have to design a forward primer consisting of restriction sites (8 base), : operator region (35 base), ribosome biding site (6 base), spacer sequence ( : about 20 base) and the priming sequence (21 base) of my target gene. The : total lenth would be 100 base for this forward primer and the reverse primer : is just 30 base. Does anyone know if this primer set would be okay for PCR : amplifying my target gene (around 720bp)? : Thanks a lot.
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Z******5 发帖数: 435 | 9 没试过。不过不太建议这么做,合成100bp以上的引物应该不便宜吧。
要是我的话,就直接在primer上加上酶切位点和保护碱基,做普通的PCR。另一部分直
接合成两条Oligo退火(参考构建shRNA),然后与酶切后的PCR产物,酶切后的载体一
起去做链接,一次就能搞定。 |
j****x 发帖数: 1704 | |
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e****s 发帖数: 1125 | 11 primer dimers!
你这个100 bp的用Primer dimers最合适了。
定2个50-60左右的primer,15bp 左右的overlapping区域,先overlapping PCR做成100
bp左右的primer.
,
primer
PCR
【在 x********u 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have to design a forward primer consisting of restriction sites (8 base), : operator region (35 base), ribosome biding site (6 base), spacer sequence ( : about 20 base) and the priming sequence (21 base) of my target gene. The : total lenth would be 100 base for this forward primer and the reverse primer : is just 30 base. Does anyone know if this primer set would be okay for PCR : amplifying my target gene (around 720bp)? : Thanks a lot.
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x********u 发帖数: 430 | 12 Thanks for all your suggestions.
I will try overlap PCR first and see what I can get. |
D**A 发帖数: 311 | 13 可以,我用过75bp的,直接SITE DIRECTED MUTAGENESIS,插入5个MUTATION. |
a*****y 发帖数: 277 | 14 PCR should work but your primers will be expensive enough that you'd better
to get the gene chemically synthesized. 720 bp = 300 USD? |
D**A 发帖数: 311 | |
X******n 发帖数: 914 | 16 做过120bp的primers,没啥问题。但是价格很贵,360刀一对。 |
s********8 发帖数: 619 | 17 没看懂这个,是把两个短的primer用PCR合成一个长的primer,再用这个长primer去做
PCR吗?
100
【在 e****s 的大作中提到】![](/moin_static193/solenoid/img/up.png) : primer dimers! : 你这个100 bp的用Primer dimers最合适了。 : 定2个50-60左右的primer,15bp 左右的overlapping区域,先overlapping PCR做成100 : bp左右的primer. : : , : primer : PCR
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d****i 发帖数: 2346 | 18 早些时候做突变用过78个的还是87个的忘了。。。 |
W****7 发帖数: 426 | 19 我做过100多+20多的,直接就出来,没什么问题,做生物实验要看运气啦!
就想艳阳天MM说的生物这个鬼东西,一切皆有可能!
即使出不来,自己多摸索一下吧,我个人认为摸索过程中的经验是最重要的。
高手之所以能成为高手都是踩着失败一路爬上来的。
版上的高手我敢说绝大部分都是摸索出来,不是别人教出来!
不信你问问艳阳天mm.......呵呵。。。。 |