a*****g
发帖数: 543 | 1 挺好的。 其它几个web-based的software（ie. Roche Universal library design and
IDT PrimerQuest) 大都基于primer3。 |
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b****r
发帖数: 17995 | 2 这种效率也不高啊。一般就是每一两天出来两三个需要设计引物的位点要设计引物
而且人基因组太复杂，很容易出现非特异性扩增。你看primer3 考虑了多少因素，随机
选20bp那样效果肯定要打很多折扣，如果10个里面有一个因此要重复的话，那一个就要
多耽误好几天，不光说成本，光是耽误的时间对于临床检测可以说很致命，还不如现在
这样多花几十分钟手工设计算了
我觉得其实就是写个script，从UCSC网页帮你输入需要的位点，再扒下来标记好的SNP
，repeat，homopolymer等要避免的地方，然后帮你扔到primer3里去，再把primer3设
计的引物扒下来，这样应该是最理想的。我觉得每个临床遗传实验室都会需要这样的软
件的，别说引用文章里，哪怕要license fee都非常值得，起码我的lab会愿意掏钱买 |
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X***n
发帖数: 366 | 3 Primer3 or Primer-Blast, the latter actually is just using primer3 and BLAST
. |
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X***n
发帖数: 366 | 4 install local primer3,
install bioperl,
run primer3 using bioperl script. |
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s******e
发帖数: 163 | 5 用命令行的 primer3
Primer3 picks primers for PCR reactions, considering as criteria:
o oligonucleotide melting temperature, size, GC content, and primer-dimer
possibilities, |
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d****o
发帖数: 361 | 6 From which company did you order your primer?
we order from Invitrogen, and they use different salt concentration
to calculate Tm. It's not about right or wrong, coz Tm changes with
oligonucleotide and salt concentrations, so u'd better calculate it by
yourself. Some softwares like Vector NTI can do it, I think Primer3
works too.
we use oligo conc. at 250 pM and salt conc. at 250 pM to calculate
the Tms. |
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f*****a
发帖数: 7 | 7 I only use SYBR. What I did is
1. Use Primer Express 1.5 or 2.0 from ABI to design primers. Follow their
guidelines. Or just use Primer3.
2. Use Amply 1.2 (or higher) and Netprimer http://www.premierbiosoft.com/n
etprimer/) to evaluate candidates.
3. Blast your primers.
4. Pick up 2-3 pairs. Do standard and dissociation curves. Chose the one wor
k best.
Make sure to chose the right normalization primers for your experiment. 18S,
actin, GAPDH, tubulin... |
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h*********9
发帖数: 361 | 8 版上时不时就有人上来问克隆问题，我就简单说一下个人的一点点经验：
克隆的第一步一般都是从PCR开始的。PCR关键是引物设计。推荐用免费的primer3。手
动设计注意引物一般GC含量45%-55%。长度在20-24之间。3'端最好不要有很多GC在一起
（40%即可）,以减少非特异产物。内切酶位点一般加在引物的5'端，最好有4-6个以上
保护碱基以利酶切（很多酶对此有硬性要求，详情参见NEB目录）。设计好的引物可以
到www.idtdna.com察看二级结构等引物信息。
第二步酶切（当然也可用TA克隆法）很关键，一定要酶切彻底才会有较高的连接效率，
我的经验是对大多数质粒，一个微克用NEB的酶1微升（10-20 U）/3-4小时，50微升体
系。PCR产物酶量要适度增加因为长度短末端多。难切的酶可酌量延长时间或加大酶量(
难不难切可参见NEB每个酶后面的说明：After a XXX-fold overdigestion with XhoI,
> 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5'
term |
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a****d
发帖数: 1919 | 11 primer bank @ Harvard Univ |
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k****i
发帖数: 53 | 13 NCBI primer design
primer3
Primer premier
DNAStar
invitrogen 在线设计 |
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A******d
发帖数: 571 | 14 同样的primer用primer3 和 primerblast算下来居然差10度。该相信哪个？要用来做
real time
pcr |
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l********5
发帖数: 52 | 15 is anyone familiar with bioperl and eprimer3, need help to make a perl
module to run primer3. |
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h*********g
发帖数: 18 | 21 I will design primers for qPCR.
First, I will find the cDNA for the gene from ensemble website. Then put the
cDNA sequence into Primer3 website.
For the parameters, I set as following
Primer size Min 20, Opt 22, Max 27,
Primer Tm, Min 57, Opt 60, Max 63.
GC% Min47, Opt 50, Max 65
It that all I need to consider? Is there anything else I need to pay
attention?
If anyone knows any good websites teaching how to design primers, please let
me know.
Thank you very much. |
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g*****n
发帖数: 250 | 22 You don't have to worry about the primers given by Primer3 or other online
programs. All the requirements for PCR primers are taken care of. Basically,
more or less 20 nt, Tm 60, GC 50%, no >4 repeats (i.g, AAAAA, GGGG, GCGCGC)
. What you really need to think about is the characteristics of the gene
that you want to test. For example, whether there are alternative spliced
variants and conserved regions with other genes of the same family. |
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h******y
发帖数: 351 | 23 http://genome.ucsc.edu/cgi-bin/hgBlat?command=start
Put your sequence in FASTA format, for example
>primer1
AGCTACAGCTACTAGCATCGACTGCGATG
>Primer2
ATGACTAGCTGGATAGCTAGCTACGATCA
>primer3
...
Make sure the primer sequence is longer than 20nt. So far this is the faste
st and most efficienct way I know. You can easily find out where the primer
s locate and whether there is any non-specific binding sites.
For pimer sequence shorter than 20nt, create four sequences by adding [AGCT]
to make it to 20n... 阅读全帖 |
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b****r
发帖数: 17995 | 24 如果能解决俺这个问题，五黄包答谢，困扰已久。不同方法越多越好，包子很多
我们做各种突变检查多态分析，经常要把目的位置（经常是1bp，最多几十bp）用PCR扩
增出来然后测序，但是目前总是只能用笨办法，先用UCSC标记好附近的SNP和repeat区
域，然后把周围几百bp拿出来扔到Primer3去设计primer，效率太低了，特别是有时候
要测几十个突变，能搞几天
我总觉得这种工作做遗传的人会经常要做，但是似乎搜不到这样的软件或者数据库提供
自动的引物设计。我觉得这样的软件相关引用率应该会非常高啊，而且不应该很难实现 |
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i*e
发帖数: 352 | 25 没有啥能100%万无一失的，BatchPD那个估计common SNPs都已经考虑了，rare的不清楚
，不过rare的话碰到的概率也小很多。此外individual个体还可能有未知的rare
variants，这个就更控制不了了。
看你之前所说，感觉是用来做validation不是detection的，所以万一有那么几个
readout不一致，换对引物或者用其他方法再验证一下。
我有可能记错，不错homopolymer和repeats在exons没有在3‘或者5’端频繁，有碰到
再分别单个手工设计引物好了。
我自己那个script，主要针对自己的实验目的，是WGS的variant validation，主要是
SNVs和小的Indels，也不单单是exonic，所以大多数面向exons的predesigned primers
就无用了，再说SNPs也没有被先排除。
设计引物的时候，input是VCF，已知的common和rare SNPs （dbSNP142）都有先
flagged，然后primer3的结果只取第一个output，之后in silico PCR再过一次，没过
的重新滚... 阅读全帖 |
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b****r
发帖数: 17995 | 26 看你什么目的，一般的不是都用primer3吗，我都用了十多年了 |
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发帖数: 1 | 28 I would try primer blast from NCBI. It's basically using PRIMER3 to design
primer followed by blast alignment to evaluate specificity.
Paste the sequence of the species-of-interest in the PCR template. In the "
Primer Pair Specificity Checking Parameters" section below, specify the
species you want to avoid the primer to amplify. In this way, the algorithm
will avoid primers having potentials on that species. |
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