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#1. Site Directed Mutagenesis by PCR - Addgene Blog
Site directed mutagenesis is a highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions) in a ...
#2. Simple and efficient site-directed mutagenesis using two ...
In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a ...
#3. Site Directed Mutagenesis | NEB
Site -directed mutagenesis (SDM) is a method to create specific, targeted changes in double stranded plasmid DNA. There are many reasons to make specific DNA ...
#4. Methods for Site-Directed Mutagenesis | IDT - Integrated DNA ...
When PCR is used for site-directed mutagenesis, the primers are designed to include the desired change, which could be base substitution, ...
#5. A high-efficiency method for site-directed mutagenesis of large ...
One of the simplest methods for site-directed mutagenesis is the method based on PCR with a pair of complementary primers (a double-stranded DNA ...
#6. Site‐Directed Mutagenesis by Polymerase Chain Reaction
The site‐directed mutagenesis using PCR has been used in molecular biology to modify gene sequences. Methods described here have allowed the introduction of ...
#7. PCR-Based Site-Directed Mutagenesis | SpringerLink
Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been ...
#8. An easy-to-use site-directed mutagenesis method with ... - NCBI
由 B Zhang 著作 · 2009 · 被引用 22 次 — Site-directed mutagenesis (SDM) has a variety of applications and is extensively used in molecular biology. ... The SDM techniques can be grouped into two major ...
#9. Site-Directed Mutagenesis | Thermo Fisher Scientific - TW
Site -directed mutagenesis is a powerful research tool used to study protein function, identify enzyme active sites, and design novel proteins in drug ...
#10. Chapter Ninteen - Site-Directed Mutagenesis - ScienceDirect ...
Site -directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study ...
#11. A simple and efficient method for in vitro site-directed ... - bioRxiv
We have developed an alternative cloning method to perform site-directed mutagenesis based on a two PCR-round procedure, followed by ligation of ...
#12. TOOLSite-Directed Mutagenesis Kit - 圖爾思公司-最強最專業 ...
Due to the efficient seamless splicing of the two PCR products by Toolsitase technology, the kit can complete the two separate sites mutation by single ...
#13. Site-directed mutagenesis - Wikipedia
The exponential amplification in PCR produces a fragment containing the desired mutation in sufficient quantity ...
#14. PCR-Mediated Site-Directed Mutagenesis - CSH Protocols
Unlike traditional site-directed mutagenesis, this protocol requires only a single PCR step using full plasmid amplification to generate point mutants.
#15. Rapid and Error-Free Site-Directed Mutagenesis by a PCR ...
Site -directed mutagenesis on large-size DNA templates is known to be very problematic because the PCR-based methods tend to introduce ...
#16. High Efficiency of Site-Directed Mutagenesis Mediated by a ...
Abstract. We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which ...
#17. Site-Directed Mutagenesis Using PCR-Mediated Introduction ...
Benchmarks. Site-Directed Mutagenesis. Using PCR-Mediated. Introduction of Silent. Mutations. BioTechniques 25:184-188 (August 1998).
#18. PCR-Based Site-Directed Mutagenesis - Springer Nature ...
Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been ...
#19. Site Directed Mutagenesis | PCR Biosystems
Site directed mutagenesis is an in vitro method for introducing specific and intentional mutations into DNA fragments. It is often performed using PCR-based ...
#20. QuikChange Site-Directed Mutagenesis Kit - Agilent
Ethanol precipitate the Dpn I digested PCR product, and resuspend in a decreased volume of water before transformation. Low mutagenesis efficiency or low colony ...
#21. Site-Directed Mutagenesis | Şen Lab
Site -Directed Mutagenesis. by Tannon Yu. Contents. BACKGROUND; REQUIRED MATERIALS; PROTOCOL. PCR amplification; DpnI digestion; High- ...
#22. Designing primers for site-directed mutagenesis - Takara Bio
To perform mutagenesis, design your PCR primers so that they have a 15-bp overlap with each other at their 5' ends and incorporate the mutation ...
#23. Single-Primer PCR for Site-Directed Mutagenesis
Objectives: To introduce site-directed mutagenesis into the pcDNA3.1(+)-F plasmid containing respiratory syncytial virus F gene coding sequence ...
#24. PCR Site-Directed Mutagenesis | DNASTAR
Site -directed mutagenesis is a molecular biology method that is used to make specific changes to the DNA sequence of a gene. In Lasergene, this workflow ...
#25. Site Directed Mutagenesis Protocol - iGEM 2014
Keep in mind that the product of the reaction is never used as a template. This mean that this is a linear amplification technique, unlike standard PCR where ...
#26. 3 Designing Primers for Site-Directed Mutagenesis - De Gruyter
In this handout you will review the basics of primer design while in the next handout you will learn about PCR amplification in practice. 3.2 Mini Project ...
#27. Q5 Site Directed Mutagenesis Kit - New England Biolabs GmbH
Site -directed mutagenesis (SDM) is a method to create specific, ... strains and incorporate mutations into the plasmid by inverse PCR with standard primers.
#28. Efficient method for site-directed mutagenesis in large ... - PLOS
Basic URMAC requires two PCR reactions, each followed by a ligation reaction to circularize the product, with an optional third enrichment PCR ...
#29. Rapid Site-Directed Mutagenesis Using Two-PCR-Generated ...
We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective ...
#30. Site-Directed Mutagenesis for In Vitro and In Vivo Experiments ...
This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, ...
#31. Instruction manual KOD -Plus- Mutagenesis Kit 0801 - TOYOBO
This kit is an inverse PCR (iPCR)-based site-directed mutagenesis kit using KOD DNA polymerase1) 2) as a high-fidelity PCR enzyme.
#32. Site-directed mutagenesis - OpenWetWare
Any highly competent cells should be ok. Stratagene recommends trying various concentrations of template DNA in the PCR step. It IS NOT PCR people!Smoore 18:52, ...
#33. Troubleshooting the Single-step PCR Site-directed ...
Troubleshooting the Single-step PCR Site-directed Mutagenesis. Procedure Intended to Create a Non-functional rop Gene in the. pBR322 Plasmid. Lina Jew.
#34. PCR mutagenesis — The Open Lab Book v1.0
PCR mutagenesis is a method for generating site-directed mutagenesis. This method can generate mutations (base substitutions, insertions, and deletions) ...
#35. (PDF) Site-Directed Mutagenesis Using the Megaprimer Method
The megaprimer method utilizes two external oligonucleotide primers and one internal mutagenic primer in two rounds of PCR with a DNA template that contains the ...
#36. A rapid and efficient PCR-based mutagenesis method ...
Major strategies of PCR-based mutagenesis include base substitution, deletion, insertion, chimeric gene generation, multiple-site mutagenesis, and random ...
#37. Enabling Fast, Efficient, and High Fidelity Site-Directed ...
Site -directed mutagenesis methods such as the QuikChange method have a number of advantages over PCR-based approaches.
#38. PrimerX -- Automated design of mutagenic primers for site ...
Design PCR primers for site-directed mutagenesis using DNA or protein sequences. Highlights: PrimerX is a web-based program written to ...
#39. Site-directed mutagenesis in Arabidopsis using custom ...
PCR using primers flanking the ZFN recognition sequence was performed on genomic DNA extracted from cells treated with ZFNs. PCR products were cloned into pCR- ...
#40. Phusion-site-directed-mutagenesis-kit-Westburg.pdf
This kit uses the highly processive Thermo Scientific Phusion Hot Start. II High-Fidelity DNA Polymerase for exponential PCR amplification of. dsDNA plasmid to ...
#41. Site-directed Mutagenesis | Universität Tübingen
Through a simple PCR reaction with overlapping, complementary oligonucleotide pairs virtually any desired mutation can be introduced in a plasmid-borne gene.
#42. Site-Directed Mutagenesis Tips and Tricks - Bitesize Bio
Site -directed mutagenesis (SDM) is a technique used to mutate one or more bases within a plasmid. This approach can change amino acid ...
#43. Site-Directed Mutagenesis (Stratagene protocol) | McManus Lab
PCR Program · 95 degrees for 1 minute · 95 degrees for 50 seconds, 60 degrees for 50 seconds, 68 degrees for 1 minute/kb of plasmid length -- repeat this step 17 ...
#44. A Novel PCR Site-Directed Mutagenesis to Modify Structure of ...
Method: the one-step PCR site-directed mutagenesis strategy can introduce mutation of gene through 5 / -end of primer, which was applied on peptide Erabutoxin B ( ...
#45. Q5® Site-Directed Mutagenesis Kit - BIOKÉ
After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and ...
#46. PickMutant™ Site-directed Mutagenesis Kit | Canvax Biotech
PickMutant™ is a reliable, robust and highly efficient PCR-based mutagenesis kit. that allows creating single or multiple point mutations, deletions or ...
#47. Efficient procedure for site-directed mutagenesis mediated by ...
This strategy is based on performing PCR reactions to create a new restriction site in the sequence of origin, corresponding to the desired mutation. The choice ...
#48. 1.3: Designing Primers for Site-Directed Mutagenesis
PCR amplification means that we synthesize (make) many copies of our DNA of interest (the coding region for a protein ...
#49. Gibson Assembly® Site-Directed Mutagenesis Kit Instructions
You may need to optimize PCR amplification reactions when using PCR primers with long homologous overlap regions. Mutagenesis Primer Design. 1. Optimally, ...
#50. US5556747A - Method for site-directed mutagenesis - Google ...
A method for site-directed mutagenesis using a third mutagenic primer in a polymerase chain reaction (PCR) based methodology.
#51. Site directed mutagenesis - SlideShare
The PCR-based mutagenesis technique commonly employed is depicted in First the target DNA. Site-directed mutagenesis is used to generate ...
#52. Site Directed Mutagenesis, Troubleshooting & FAQs - Assay ...
Site Directed Mutagenesis Troubleshooting & FAQs ; Incorrect site-directed mutation. The primers are not designed correctly. Check the primer design. ; The ...
#53. Thermo Scientific Phusion Site-Directed Mutagenesis Kit
Gene Editing and Gene Synthesis Tools · Home · Products · PCR Equipment and Supplies · PCR Supplies · High Fidelity PCR Reagents and Kits · F541.
#54. GENEWIZ from Azenta | Site-Directed Mutagenesis
... site-directed mutagenesis projects efficiently and cost-effectively. ... High-fidelity enzymes to minimize PCR errors and optimized proprietary GENEWIZ ...
#55. GeNeiTM InSitu PCR-Based Site Directed Mutagenesis Kit, 10 ...
InSitu PCR-Based Site Directed Mutagenesis Kit is a fast, easy and efficient PCR based method to introduce point mutations as well as insertion and deletion ...
#56. Q5® Site-Directed Mutagenesis (E0554) - Protocols.io
The Q5® Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure ...
#57. UNIT IX Site-directed Mutagenesis (SDM) - Shivaji College
B. Oligonucleotide-Directed Mutagenesis with Plasmid DNA. C. PCR based mutagenesis i. PCR-Amplified Oligonucleotide-Directed. Mutagenesis.
#58. Site-Directed Mutagenesis Services - GenScript
Create specific DNA mutations with GenScript site-directed mutagenesis services, starting at only $99/mutation with 100% accuracy guaranteed on PCR ...
#59. Thermo Scientific Phusion Site-Directed Mutagenesis Kit
This kit uses the highly processive Thermo. Scientific Phusion Hot Start II High-Fidelity DNA Polymerase for exponential PCR amplification of. dsDNA plasmid to ...
#60. CRISPR-Directed Gene Editing Catalyzes Precise Gene ...
A PCR-based site-directed mutagenesis technique is often used to engineer these variants. While these tools are efficient, ...
#61. Site-directed Mutagenesis using PCR-第五章文章资源-仲恺农业工程 ...
Site -directed Mutagenesis using PCR. Michael P. Weiner, Tim Gackstetter, Gina L. Costa, John C. Bauer, and Keith A. Kretz ...
#62. Random Mutagenesis by Insertion of Error-Prone PCR ...
Site -Directed Mutagenesis. Each mutation was introduced into wildtype Pcry3A-mpd expression cassette through overlap PCR (Shevchuk et al., ...
#63. NEB Q5 Site-Directed Mutagenesis Kit - LabJot
Ukierunkowaną mutagenezę przeprowadza się w oparciu o reakcję PCR, w której użyta jest polimeraza wysokiej wierności – Q5 Hot Start High-Fidelity DNA Polymerase ...
#64. [試劑耗材]這就是點突變(Site-Directed Mutagenesis)
威健生技部落格搬到新家嘍! 目前舊的痞客邦已不再更新, 同文章請移至新網址-> [試劑耗材]這就是點突變(Site-Directed Mutagenesis) - 點突變小歷.
#65. [求救] site-directed mutagenesis - 精華區Biotech - 批踢踢實業坊
曾想過把多管PCR產物跑膠(先以DpnI digest)後，把少量的產物集中 ... 若只初相見) 看板: Biotech 標題: Re: [求救] site-directed mutagenesis 時間: ...
#66. Site Directed Mutagenesis - ppt video online download
Site Directed Mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule known as a plasmid.
#67. What is site directed mutagenesis PDF?
Site -directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene ...
#68. site-directed mutagenesis protocol | Differbetween
When PCR is used for site-directed mutagenesis, the primers are designed to include the ...
#69. Site-Directed Mutagenesis Protocol using PCR - Wnt Proteins
Site -Directed Mutagenesis Protocol using PCR. Design primer pair so that the mutation (s) are near the middle of the primer, and the primer has a melting ...
#70. Cigarette smoking is a secondary cause of folliculin loss | Thorax
QuikChange II XL site-directed mutagenesis kits (Cat#: 200522) were ... Quantitative PCR (qPCR) experiments were performed using the ...
#71. PCR Cloning Protocols - 第 174 頁 - Google 圖書結果
We have briefly discussed some basic PCR approaches to DNA mutagenesis and ... L. R. (1989) Site-directed mutagenesis by overlap extension using the PCR.
#72. Principles and Technical Aspects of PCR Amplification
Mutagenesis. by. PCR. The selective introduction of nucleotide changes (mutations) within a known DNA sequence is a process called site-directed mutagenesis ...
#73. Allergy Bioinformatics - 第 209 頁 - Google 圖書結果
Site -directed mutagenesis can introduce aimed changes to DNA fragments (including ... Error-prone PCR is performed under artificially controlled conditions ...
#74. DNA Cloning: A Hands-on Approach - 第 60 頁 - Google 圖書結果
Since site-directed mutagenesis uses PCR, there is always the possibility that undesirable mutations can be introduced. This means that the larger the ...
#75. Comprehensive Biotechnology - 第 524 頁 - Google 圖書結果
SITE - DIRECTED MUTAGENESIS USING PCR In vitro site - directed mutagenesis is an invaluable technique for studying protein structurefunction relationships ...
#76. Site-directed Mutagenesis 定点突变两种方法的原理~_哔哩哔哩
#77. Single-molecule Taq DNA polymerase dynamics - Science
At PCR temperatures, the full recordings contained thousands of catalytic ... Q5 site-directed mutagenesis was applied according to the ...
#78. A novel synonymous KMT2B variant in a patient with dystonia ...
... variant was then introduced through site-directed mutagenesis. ... Sanger sequencing of the PCR products revealed that c.5073C>T caused ...
#79. Multisystem inflammatory syndrome as a late complication in ...
... by reverse transcription-polymerase chain reaction (RT-PCR). ... to DOCK8 mutant sequences were produced by site-directed mutagenesis ...
#80. Palzkill Lab Research Projects - Baylor College of Medicine
The lab uses a combination of site-directed mutagenesis, ... Current diagnostics--RT-PCR and enzyme immunoassays--are severely limited for use at the ...
#81. NGS Barcodes & UPS Labels & NGS Coupons - Eurofins ...
With one Coupon you get: Amplicon generation using 2-step PCR; MiSeq Sequencing; 60k read pairs (2x300 bp). Available for:.
#82. Lambda Red-mediated recombinogenic engineering ... - CORE
and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, ... oligo-directed gene replacements that yeast geneticists.
#83. Member List - ERNEST - Cost Action
Name Country WG Techniques of ex... Cordomí Arnau; Universitat Autònoma de Barcelona ES WG1, WG3 Structural; Chemi... Dr. Daniel Hilger; Philipps‑Universität Marburg DE WG1, WG3 Biophysical; Bioc... Gmeiner, Peter; FAU; Website DE WG3 Chemistry; Pharm...
#84. Fundamental Molecular Biology - 第 592 頁 - Google 圖書結果
... element of interest has been modified by in vitro mutagenesis (Tool Box 14.2). (a) Deletion Mutagenesis +1 (c) Site-Directed Mutagenesis by PCR 2 ...
#85. Microbial Genetics - 第 360 頁 - Google 圖書結果
Hence, PCR can be a powerful tool to introduce mismatches in a linear DNA fragment. This has been utilized for creating site-directed mutagenesis in a rapid ...
#86. Molecular Biology of the Toxic Response - 第 19 頁 - Google 圖書結果
The megaprimer method employs two subsequent PCR reactions . ... Site - directed mutagenesis of the TGF - β2 CRE / ATF site has generated results similar to ...
#87. MUTANT ß-GLUCOSIDASE - Patent Details - QuickCompany
49 分鐘前 — Alternatively, site-specific mutagenesis into the parent BGL gene can be performed by SOE (splicing by overflow extension) -PCR method (Horton ...
site-directed mutagenesis pcr 在 [求救] site-directed mutagenesis - 精華區Biotech - 批踢踢實業坊 的推薦與評價
我現在是做A基因的點突變，
用的方法是利用complement primer 25bp，而突變設計在中間
利用這組primer做完pcDNA3-A一整圈plasmid，大小約6.7kb
目前卡在PCR出來的產物很少很少，以致於做transform後都沒有colony長出來…
曾想過把多管PCR產物跑膠(先以DpnI digest)後，把少量的產物集中一起elution
但濃度還是很低(1.1 ng/ul)。
另外因此突變點位在基因末端(離stop codon約30bp,全長約1.4kb)，所以用下述方法
心想應該行不通
F1----------> F2--------->
------------------------------------------------------
||||||||||||||||||||||||||||||||||||||||||||||||||||||
------------------------------------------------------
R1<---------
R2<----------
二段PCR產物overlap處為mutation site，不過會變成F1、R1夾出來1300多bp而F2、R2
夾出來可能100bp不到的產物，再將二個大小差距那麼大的產物加在一起覺得不太可行。
所以想請問版上有人遇過這種狀況嗎？
Primer重新order過了，Tm值60.XX，PCR用Phusion polymerase。
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◆ From: 140.114.202.163
※ 編輯: nhxcyge 來自: 140.114.202.163 (11/11 21:24)
> -------------------------------------------------------------------------- <
作者: tutj (人生若只初相見) 看板: Biotech
標題: Re: [求救] site-directed mutagenesis
時間: Sat Nov 12 14:45:20 2011
※ 引述《nhxcyge (人生就像個不可逆反應)》之銘言：
: 我現在是做A基因的點突變，
: 用的方法是利用complement primer 25bp，而突變設計在中間
complement primer應該是stratagene QuikChange的方法吧？
: 利用這組primer做完pcDNA3-A一整圈plasmid，大小約6.7kb
: 目前卡在PCR出來的產物很少很少，以致於做transform後都沒有colony長出來…
: 曾想過把多管PCR產物跑膠(先以DpnI digest)後，把少量的產物集中一起elution
: 但濃度還是很低(1.1 ng/ul)。
: 另外因此突變點位在基因末端(離stop codon約30bp,全長約1.4kb)，所以用下述方法
: 心想應該行不通
: F1----------> F2--------->
: ------------------------------------------------------
: ||||||||||||||||||||||||||||||||||||||||||||||||||||||
: ------------------------------------------------------
: R1<---------
: R2<----------
: 二段PCR產物overlap處為mutation site，不過會變成F1、R1夾出來1300多bp而F2、R2
: 夾出來可能100bp不到的產物，再將二個大小差距那麼大的產物加在一起覺得不太可行。
: 所以想請問版上有人遇過這種狀況嗎？
: Primer重新order過了，Tm值60.XX，PCR用Phusion polymerase。
你的Tm值是照說明書的公式算的嗎？
我有找到一個網站可以幫忙算
https://depts.washington.edu/bakerpg/primertemp/primertemp.html
如果你是照公式算, 說明書要求至少要78℃以上; 如果不是, 那Tm值一定太低
這方法Tm值一定要夠高。因為primer完全complement, 還有突變的base
所以primer間的Tm值會比primer對DNA template的Tm值高
如果一開始設計的Tm不夠高
PCR時primer會傾向形成primer dimer而不是黏上DNA template
至於你說的PCR產量低, 我之前有找到一篇paper
裡面說QuikChange有幾個缺點。 一是上面提的易形成primer dimer
二是不能同時多點突變; 三就是PCR產量低。
因為PCR從primer順著plasmid跑完一圈後頭尾接不起來形成nick
一對primer又是完全complement
整個PCR program只有parental plasmid可以做為DNA template
那篇paper發明了一種新方法
F1 ---------m----------->
------------------*----------------------------------- *: nick
|||||||||||||||||||||||||||||||||||||||||||||||||||||| m: mutation
-----------------------------------*------------------
R1<-----------------m------
如圖, primer間只有部份overlap, mutation site在overlap region中間
3'延伸出去的部份蓋過PCR產物的nick
所以新生成帶有mutation的DNA可以當成template進刪PCR reaction
故一開始需要加入的DNA template可以降低, 提高之後挑mutant colony的機率
paper現在不在手邊, 如果你有興趣等星期一去實驗室我可以提供
根據我整理的protocol
這邊primer跟template之間的Tm值稱為Tm no, primer-primer間的Tm稱為Tm pp
設計primer時Tm no要比Tm pp高5~10℃
PCR reaction的配方: 1x PCR reaction buffer, 1 uM primer pair,
2~10 ng DNA template (我用5 ng), 0.2 mM each dNTP (我怕不夠加到0.4 mM)
3u Pfu polymerase (其他DNA polymerase應該也可以), 補水到50 ul
PCR program:
1. 95℃ 5 min x1 cycle
2. 95℃ 1 min (cycle數我照QuikChange的建議, 換掉a.a.跑16 cycles)
Tm no-5℃ 1 min
72℃ 1 kp/2 min
3. 95℃ 1 min x1 cycle
Tm pp-5℃ 1 min
72℃ 30min (據說此cycle能增加完整plasmid的生成率)
elongation因為我用PfuUlatr II, 所以減到1 kb/min, 跑68℃
然後primer因為高GC% Tm值超高,減五度也超過65℃
所以annealing照QuikChange跑55℃ 最後一個cycle省略成只跑72℃ 30min
取10 ul PCR product跑膠有超亮的single band
提供給你參考~
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◆ From: 175.181.106.80
> -------------------------------------------------------------------------- <
作者: blence ( ) 站內: Biotech
標題: Re: [求救] site-directed mutagenesis
時間: Sun Nov 13 00:23:39 2011
補充一下
QuickChange是可以同時做多點突變，甚至做1kb以上大片段deletion的
不過我同意將原本protocol改成用partial complement primer效率會更好
只不過我的經驗是primer至少要有18~20bp以上的complement
幾次complement太短(嘗試<15bp)反而都失敗
因此如果只是1~2bp的mutation，primer大概25bp都ok
也就是
(3bp)( 20bp )(3bp)
Forward: -----M------------>
Reverse: <---------M--------
primer太長，Tm就會偏高，短一點的話，GC高的部分會比較好做
如果再搭配plasmid 50ng
對於1~2bp極短片段的突變幾乎用55度萬用Tm應該不成問題
: PCR時primer會傾向形成primer dimer而不是黏上DNA template
: 至於你說的PCR產量低, 我之前有找到一篇paper
: 裡面說QuikChange有幾個缺點。 一是上面提的易形成primer dimer
: 二是不能同時多點突變; 三就是PCR產量低。
: 因為PCR從primer順著plasmid跑完一圈後頭尾接不起來形成nick
: 一對primer又是完全complement
: 整個PCR program只有parental plasmid可以做為DNA template
: 那篇paper發明了一種新方法
: F1 ---------m----------->
: ------------------*----------------------------------- *: nick
: |||||||||||||||||||||||||||||||||||||||||||||||||||||| m: mutation
: -----------------------------------*------------------
: R1<-----------------m------
: 如圖, primer間只有部份overlap, mutation site在overlap region中間
: 3'延伸出去的部份蓋過PCR產物的nick
: 所以新生成帶有mutation的DNA可以當成template進刪PCR reaction
我想新生成的含m的DNA應該是不能當成template的
否則最後的產物會是一堆兩端都含有m的blunt end product
（primer已經有m了，新生成的也有m）
也就是
(F1 primer) ( R1 novel synthesis )
-----m-----------------------------------m---->
以及 ( F1 novel synthesis ) (R1 primer)
<-----m-----------------------------------m----
: 故一開始需要加入的DNA template可以降低, 提高之後挑mutant colony的機率
: paper現在不在手邊, 如果你有興趣等星期一去實驗室我可以提供
: 根據我整理的protocol
: 這邊primer跟template之間的Tm值稱為Tm no, primer-primer間的Tm稱為Tm pp
: 設計primer時Tm no要比Tm pp高5~10℃
: PCR reaction的配方: 1x PCR reaction buffer, 1 uM primer pair,
: 2~10 ng DNA template (我用5 ng), 0.2 mM each dNTP (我怕不夠加到0.4 mM)
: 3u Pfu polymerase (其他DNA polymerase應該也可以), 補水到50 ul
: PCR program:
: 1. 95℃ 5 min x1 cycle
: 2. 95℃ 1 min (cycle數我照QuikChange的建議, 換掉a.a.跑16 cycles)
: Tm no-5℃ 1 min
: 72℃ 1 kp/2 min
: 3. 95℃ 1 min x1 cycle
: Tm pp-5℃ 1 min
: 72℃ 30min (據說此cycle能增加完整plasmid的生成率)
: elongation因為我用PfuUlatr II, 所以減到1 kb/min, 跑68℃
: 然後primer因為高GC% Tm值超高,減五度也超過65℃
: 所以annealing照QuikChange跑55℃ 最後一個cycle省略成只跑72℃ 30min
: 取10 ul PCR product跑膠有超亮的single band
: 提供給你參考~
我認識的大多跑10~25ul reaction
因此都是用3~5ul check
另外一些method paper提過extension 68度會比72度有降低突變的機率
不過缺點是amphify效率差
product不用多，一般EtBr的limitation約10ng左右
只要ErBr看得到，那失敗的探討就可能不是QuickChange的過程
※ 編輯: blence 來自: 140.109.40.29 (11/13 17:00)
> -------------------------------------------------------------------------- <
作者: aggaci (台南工作的基隆人) 看板: Biotech
標題: Re: [求救] site-directed mutagenesis
時間: Wed Nov 16 18:11:23 2011
※ 引述《aggaci (台南工作的基隆人)》之銘言：
※ 引述《nhxcyge (人生就像個不可逆反應)》之銘言：
: 我現在是做A基因的點突變，
: 用的方法是利用complement primer 25bp，而突變設計在中間
: 利用這組primer做完pcDNA3-A一整圈plasmid，大小約6.7kb
: 目前卡在PCR出來的產物很少很少，以致於做transform後都沒有colony長出來…
: 曾想過把多管PCR產物跑膠(先以DpnI digest)後，把少量的產物集中一起elution
: 但濃度還是很低(1.1 ng/ul)。
: 另外因此突變點位在基因末端(離stop codon約30bp,全長約1.4kb)，所以用下述方法
: 心想應該行不通
: F1----------> F2--------->
: ------------------------------------------------------
: ||||||||||||||||||||||||||||||||||||||||||||||||||||||
: ------------------------------------------------------
: R1<---------
: R2<----------
: 二段PCR產物overlap處為mutation site，不過會變成F1、R1夾出來1300多bp而F2、R2
: 夾出來可能100bp不到的產物，再將二個大小差距那麼大的產物加在一起覺得不太可行。
: 所以想請問版上有人遇過這種狀況嗎？
: Primer重新order過了，Tm值60.XX，PCR用Phusion polymerase。
我的方法不一樣 提供給大家
我的是
P---------m----------->
------------------------------------------------------ m:mutation
||||||||||||||||||||||||||||||||||||||||||||||||||||||
------------------------------------------------------
<---------------P
這樣做PCR 因為沒重疊所以不太會形成primer dimer 會比較好做
要注意的地方在於我的primer尾端會加phosphate
因為這樣做出來是linear DNA所以之後拿去作ligation......
效率不錯!!我覺得比傳統quickchange好做很多
另外我用的是fynzyme的phusion pfu
這pfu超好用 作的 超準 超快 超多
--
單身不代表寂寞，寂寞是走在一個不愛妳的人身邊的時候。
--
※ 發信站: 批踢踢實業坊(ptt.cc)
◆ From: 140.116.253.121
※ 編輯: aggaci 來自: 140.116.253.121 (11/15 23:41)
※ 編輯: aggaci 來自: 140.116.253.121 (11/15 23:42)
※ 編輯: aggaci 來自: 140.116.253.121 (11/15 23:43)
簡單阿
我們lab的做法是 insert 沒錯就好
之後把insert 切下來再接到新的vector上
2 step pcr難用又難做
PFU還是無法保証vector sequence有沒出錯
除非你去對整個vector定序
所以最安全的做法是 把insert 切下來再接到新的vector上
--
單身不代表寂寞，寂寞是走在一個不愛妳的人身邊的時候。
--
※ 發信站: 批踢踢實業坊(ptt.cc)
◆ From: 140.116.253.121
※ 編輯: aggaci 來自: 140.116.253.121 (11/16 18:12)
> -------------------------------------------------------------------------- <
作者: aggaci (台南工作的基隆人) 看板: Biotech
標題: Re: [求救] site-directed mutagenesis
時間: Tue Nov 15 23:41:15 2011
※ 引述《nhxcyge (人生就像個不可逆反應)》之銘言：
: 我現在是做A基因的點突變，
: 用的方法是利用complement primer 25bp，而突變設計在中間
: 利用這組primer做完pcDNA3-A一整圈plasmid，大小約6.7kb
: 目前卡在PCR出來的產物很少很少，以致於做transform後都沒有colony長出來…
: 曾想過把多管PCR產物跑膠(先以DpnI digest)後，把少量的產物集中一起elution
: 但濃度還是很低(1.1 ng/ul)。
: 另外因此突變點位在基因末端(離stop codon約30bp,全長約1.4kb)，所以用下述方法
: 心想應該行不通
: F1----------> F2--------->
: ------------------------------------------------------
: ||||||||||||||||||||||||||||||||||||||||||||||||||||||
: ------------------------------------------------------
: R1<---------
: R2<----------
: 二段PCR產物overlap處為mutation site，不過會變成F1、R1夾出來1300多bp而F2、R2
: 夾出來可能100bp不到的產物，再將二個大小差距那麼大的產物加在一起覺得不太可行。
: 所以想請問版上有人遇過這種狀況嗎？
: Primer重新order過了，Tm值60.XX，PCR用Phusion polymerase。
我的方法不一樣 提供給大家
我的是
P---------m----------->
------------------------------------------------------ m:mutation
||||||||||||||||||||||||||||||||||||||||||||||||||||||
------------------------------------------------------
<---------------P
這樣做PCR 因為沒重疊所以不太會形成primer dimer 會比較好做
要注意的地方在於我的primer尾端會加phosphate
因為這樣做出來是linear DNA所以之後拿去作ligation......
效率不錯!!我覺得比傳統quickchange好做很多
另外我用的是fynzyme的phusion pfu
這pfu超好用 作的 超準 超快 超多
--
單身不代表寂寞，寂寞是走在一個不愛妳的人身邊的時候。
--
※ 發信站: 批踢踢實業坊(ptt.cc)
◆ From: 140.116.253.121
※ 編輯: aggaci 來自: 140.116.253.121 (11/15 23:41)
※ 編輯: aggaci 來自: 140.116.253.121 (11/15 23:42)
※ 編輯: aggaci 來自: 140.116.253.121 (11/15 23:43)
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