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 JoVE General

Tüm Plazmidler Homemade Sitesi Yönetmen Mutagenez

1, 2

1Department of Biology, Johannes Gutenberg-University Mainz, Germany, 2Proteomics division, AlPlanta, Neustadt an der Weinstrasse, Germany

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    Summary

    Tüm plazmid Site directed mutagenesis özgün bir plazmid biraz farklı varyasyonlarını oluşturmak için basit bir yoludur. Burada temel yerine plazmid kullanarak standart reaktifleri tanıtmak için kolay ve maliyet etkin bir şekilde göstermektedir.

    Date Published: 5/11/2009, Issue 27; doi: 10.3791/1135

    Cite this Article

    Laible, M., Boonrod, K. Homemade Site Directed Mutagenesis of Whole Plasmids. J. Vis. Exp. (27), e1135, doi:10.3791/1135 (2009).

    Abstract

    Tüm plazmid Site directed mutagenesis özgün bir plazmid biraz farklı varyasyonlarını oluşturmak için basit bir yoludur. Bu yöntem ile klonlanmış hedef gen doğrudan bir plazmid içine birkaç üslerinden ikame, silme veya ekleme tarafından değiştirilebilir. Sadece olmayan bir PCR tabanlı thermocycling reaksiyon, tüm plazmid yükseltme çalışır. Reaksiyon sırasında istenilen mutasyonu taşıyan mutajenik primerler, yeni sentezlenmiş plazmid entegre edilmiştir. Bu video öğretici bir plazmid temel yerine tanıtmak için kolay ve maliyet etkin bir şekilde göstermektedir. Protokol standart reaktifleri ile çalışır ve genellikle çok pahalı ticari kitleri, bağımsız. Bu protokolü uygulamak bazı ticari kitleri kullanılarak maliyeti ne sekizde bir tepkimenin toplam maliyetini düşürebilir. Bu video da sürecinde kritik adımlar yorum ve mutajenik primerler dizayn konusunda ayrıntılı talimatlar vermek.

    Protocol

    Yöntemin prensibi:

    Bu site tüm plazmid mutagenez Bu video doğrudan bir plazmid içine birkaç üsleri ikamesi, silme veya ekleme klonlanmış bir hedef gen değiştirmek için izin veren bir mutagenez yöntemi açıkladı yönetti. Olmayan bir PCR tabanlı thermocycling tepki olarak, tüm plazmid yükseltme çalışır. Reaksiyon sırasında orijinal plazmid uyumsuzlukları şeklinde istenen mutasyonu taşıyan mutajenik astar, yeni sentezlenmiş plazmid entegre edilmiştir. Reaksiyon orijinal plazmid çıkarıldıktan sonra mutasyona uğramış plazmid E. dönüşmüş olur Bu yöntemin mutasyon verimliliği% 100 değildir, çünkü coli aşağıdaki adımları, sadece tarama amaçlı . Tüm prosedürü üç gün sürer, ancak ana kısmı bir gün içinde yapılabilir.

    1.0 Birinci gün:

    1.1 Thermocycling reaksiyon:

    Orijinal Plazmid: Bu site mutagenez protokolü yönettiği 10kb için plazmid ile iyi çalışır. Büyük Plazmidler bu yöntem ile mutasyona biraz zor ve biraz sabır ve thermocycling koşullar ve / veya yetkili hücreleri ayarı alabilir. Ayrıca plazmid ile çalıştıkları bir baraj + bakteri suşu izole edilmelidir. Thermocycling reaksiyon için 10 ila 60 NGS mutasyona istediğiniz plazmid gerekecektir.

    Astarlar: thermocycling tepki kurmadan önce elinizde primerler var. Her mutajenik astar yaklaşık 150 ng gerekir. 01:10 seyreltilmiş 100 pM stokunun 1.5 ul alırsak tamam. Mutajenik primerler dizayn ederken göz önünde bulundurmanız gereken birkaç basit kurallar vardır:

    • Primerler birbirini tamamlayıcı olmalıdır
    • Primerler uzunluğu 25 ve 45 nükleotidler arasında olmalıdır
    • Orijinal plazmid uyumsuzlukları şeklinde mutasyonlar her iki astarın yer almalıdır
    • Uyumsuzlukları astar merkezli ve her iki tarafta en az 8 nükleotidler tarafından çevrili olmalıdır
    • Astar en az% 40 GC-İçerik olmalıdır
    • Primerler, 5 başbakan, 3 başbakan, bir veya daha fazla Gs veya Cs ile bitmeli
    • Primerler fosforile olmak zorunda değildir, ne de FPLC veya SAYFA saflaştırılmış, sadece olmaları gerektiği desalted olmaya gerek yok.
    • Tm hesaplanması için herhangi bir formül değil, nakliye belgesi Tm 60 daha yüksek ° C olmalıdır gerekmez
    • Tarama amacıyla mutasyonu ile bir kısıtlama site eklemek veya silmek için pratiktir. Çünkü genetik kod dejenerasyon istediğiniz mutasyon ile bir kısıtlama sitesine girmek için birçok olasılık vardır. New England Biolabs web sitesi, böylesine uygun bir kısıtlama site bulmak için bir araç sağlar. DNA belirsizlik kodu kullanarak, arzu amino asit dizisi için kodlama astar dizisine girmek yeterlidir. Enzim bulucu aracı kısıtlama siteleri istediğiniz mutasyon paralel olarak tanıtıldı olabilir anlatacağım. Bu şekilde, mümkün ya da pratik herhangi bir kısıtlama sitesinde bulamazsanız, mutasyon yerinde genetik kod ile ilgili yeni bir kısıtlama olmadan herhangi bir site ekleyebilirsiniz. Bundan sonra bu kısıtlamayı sitesi yeni mutajenik astar ekleme silinecek olan mutasyonlar bir dizi şablon olarak bu plazmid kullanabilirsiniz. Bu yaklaşım, aynı yerde plazmid mutasyonların bir dizi yapmak planlıyorsanız çok kullanışlı olabilir.

    DNA Polimeraz: Bu yöntem için 3'-5 'eksonükleaz faaliyet gösteren ve künt uçları oluşturur thermostable DNA Polimeraz gerekir. Biz her zaman Fermentas rekombinant Pfu-Polimeraz kullanın. Amplifikasyon adımları ile ilgili sorunlar var ise daha yüksek bir kalite polimeraz deneyebilirsiniz, ama bir çok amaç için standart Pfu yeterli olacaktır. DNTP, polimeraz tampon ve su ile reaksiyona tamamlayın.

    1.2 Thermocycling koşulları

    PCR şu şekilde ayarlanması gerekir. Ilk ve tekrarlayan denatürasyon faz 95 ° C 30sn ayarlanır

    Primerlerin tavlama sıcaklığı, karmaşık formüller ile hesaplanır olmamalıdır. Biz genellikle, tavlama sıcaklığı 55 ° C ve bir dakika tavlama zaman ayarlayın. Çoğunlukla kullanarak 25 - 30 nükleotidlerin primerler için, bu bizim için zaman% 95 çalışır. Ve 60 ° C - Bu işe gerekir Zaten, sadece tavlama sıcaklığı 50 arasında değişen deneyin

    Uzama sıcaklığı kullanmak polimeraz bağlıdır. Bu gösteri biz bir uzama sıcaklığı 72 çağrıları Fermentas Pfu Polimeraz ° C. Uzama süresi plazmid büyüklüğüne göre değişir. Biz her zaman kb başına 1 dakika hesaplamak ve buna bir ekstra dakika eklemekzaman. Örneğin, bir 9KB'e plazmid uzama süresi olarak 10dk seçsin.

    18 döngüleri, daha sonra kullanmak için yeterli plazmid mutasyona uğramış oluşturmak için yeterlidir. Düşük döngülerinin sayısını tutmak, aynı zamanda size zaman kazandırır.

    Şekil 1

    Thermocycling reaksiyon sonra 1.3 Gelcheck

    Başarılı amplifikasyon bir elektroforez yaparak kontrol edilmelidir. Bisiklet bittikten sonra doğrudan reaksiyon 5 ul,% 1 TAE agaroz jel üzerine yerleştirin. Amplifikasyon başarılı olursa, farklı bir bant göreceksiniz. Ancak, yeni sentezlenen DNA jel üzerinde açıkça görünür değilse, bütün tepki çökelmesine denemek ve bir sonraki adım dönüşüm için kullanabilirsiniz. Bizim için bu, nadiren çalıştı ama denemeye değer. Reaksiyon işe yaramazsa, yapılacak en iyi şey, tavlama sıcaklığını ayarlamak için.

    1.4 DpnI sindirim:

    Dönüşüm reaksiyon güçlü bir arka plan önlemek için önce bir şablon olarak görev orijinal plazmid uzaklaştırılmalıdır. Bu DpnI ile sınırlama sindirim yapılır. Bu kısıtlama endonükleaz sadece plazmid metil keser. Bir metil sahipken, tanıma ve kısıtlama sitesi sırası GAİK. DpnI ile sindirerek zaman sadece bir baraj izole edildiği orijinal unmutated plazmid + suşu kesim alır, metil değil, yeni sentezlenmiş mutasyona uğramış plazmid DpnI etkilenmez. Sadece reaksiyon 1-2 ul DpnI eklemek ve en az bir saat inkübe 37 ° °. Hızlı sindirimi DpnI kullanarak kuluçka süresi yaklaşık 15 dakika azaltılabilir. DpnI ve bu kısıtlamanın sindirim inkübasyon süresi kalitesi, büyük ölçüde unmutated plazmid ile arka plan ne kadar güçlü olacaktır belirler.

    1.5 Dönüşüm:

    DpnI sindirim sonra plazmid yetkili E. dönüşebilmesi için hazır coli hücrelerinde. Sadece yetkili hücrelerin içine DpnI sindirim reaksiyon 5 ul eklemek ve laboratuarda önerildiği gibi, dönüşümü gerçekleştirmek. Bizim durumumuzda bu plazmid bakteri karışımı 30 dakika boyunca buz üzerinde inkübe ve sonra 42 ° C'de 90 sn ısı şok ısı şok yordamı kullanın. 200 ul SOC çözüm ekledikten sonra, bakteri, şiddetle, 1 saat boyunca 37 ° C sallayarak inkübe edilir 1 saat sonra bakterilerin agar ortamı seçme kaplanmıştır.

    2.0 Gün iki

    2.1 klonlar parçası 1 Eleme: klonların seçimi

    Çünkü mutasyon verimi% 100 değildir, sizin mutantlar için ekran gerekir. Biz bunu biz eklenen veya astar entegrasyon yoluyla silinen kısıtlama sitenin varlığı ya da yokluğu kontrol sınırlama sindirim. Bu amaçla genellikle sekiz koloniler pick up ve ertesi gün plazmid hazırlık için onları gece boyunca büyüyecek.

    3,0 Gün üç

    3.1 klonlar parçası 2 Tarama: Kısıtlama belirteç enzim ile sindirim

    Üçüncü gününde belirteç enzim Mini Hazırlık ve sonraki sınırlama sindirim gerçekleştirin. Jel sonra istenilen mutasyon taşıyan ve hangilerinin klonların hangisinin görebilirsiniz.

    Sınırlama sindirim kullanarak tarama yöntemi çok iyi çalışıyor. Bununla birlikte, sıralama başarılı mutasyon olduğunu teyit etmelidir.

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    Discussion

    Sitesi directed mutagenesis bir plazmid tarafından taşınan bir gen mutasyona hızlı bir şekilde sağlayan bir mutagenez yöntemdir. Tüm reaksiyon sadece bir gün içinde yapılabilir. Bu yöntemle, istenilen mutasyonları taşıyan ücretsiz primerler sadece bir çift ve Pfu-Polimeraz gibi bir redaksiyon polimeraz gerekecektir. Yeni sentezlenmiş plazmid restriksiyon enzim DpnI ile reaksiyon sindirerek ebeveyn plazmid ayrılabilir. Bu enzim sadece metillenmiş DNA sindirir. Bu nedenle sadece yeni sentezlenmiş plazmid dönüştürülebilir. Bu eğitimde ev yapımı bir mutagenez kiti kullanarak bir site directed mutagenesis performans gösterdi. Bu kitin faydası, etkinliği kalırken maliyet tasarrufu. Bu yöntemin kritik noktaları astar tasarım ve tavlama sıcaklığı. Durumda, yeni sentezlenen DNA elektroforez sonra, hangi yöntemin başarısızlık gösterebilir, bir bakteri dönüşüm için tüm reaksiyon ve kullanılması, çöktürmek için deneyebilirsiniz görüntülenmiştir olamaz. Ancak bu sorunu çözmek için en iyi yolu, tavlama sıcaklığı optimize veya primerler sabit bölgenin uzunluğunu artırmak için çalışıyor. Ayrıca, yüksek kaliteli bir polimeraz veya restriksiyon enzimi kullanarak da reaksiyon duyarlılığı artırabilir. Yetkili hücreler de, bu yöntemin successfulness etkileri önemli bir faktördür. Bu nedenle, orta (107 kob / mg DNA) veya yüksek (109 kob / mg DNA) yetkili hücreleri tercih edilir.

    Bu eğitimde ev kitini kullanarak silme veya ekleme göstermek olmasa da, biz bu laboratuvarda yapmak için başarılı olmuştur. Biz, aynı zamanda başarılı bir şekilde en az 3 üsleri, yedek eklemek veya silmek için başardık.

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    Disclosures

    Materials

    Name Company Catalog Number Comments
    Pfu polymerase (recombinant or native) Fermentas EP0571 or EP0501
    dNTP mix 10 mM Fermentas R0191
    DpnI or DpnI Fast digest Fermentas ER1701
    primers Invitrogen desalted

    References

    1. Papworth, C, Bauer, JC, Braman, J, Wright, DA QuikChange site-directed mutagenesis. Strategies 1996, 9:3-4

    Comments

    33 Comments

    I know your publication is not about a kit, but I was wondering if you can help me. Using a kit, I get through the amplification step with a product, but the transformation is unsuccessful. The control plasmid I use in every transformation (has not undergone amplification) gives positive results. I tried lengthening the extension time, yet still, no colonies. Do you have any idea what could be wrong? I've heard about duplication of primers during amplification. Do you think this could be the problem? Unfortunately, I don't have a restriction site where I need the mutation. Thank you for any help!
    Reply

    Posted by: AnonymousJuly 23, 2009, 2:11 PM

    Another reason why the transformation is not working, could be the size of your plasmid. With large plasmids we also have problems in transformation. Try using very high competent cells or even electroporation. Hope I could help you.
    Reply

    Posted by: AnonymousJuly 31, 2009, 8:04 AM

    Hi Leah, which kit are you using? DŒs it use the same principle as we use? There are other kits on the market where you have to perform ligation prior to transformation (Phusion kit from neb). You may also check if it actually is the right plasmid you are amplifiying by performing restriction digestion with it after amplification and comapre it to your input plasmid. Otherwise you could use your input plasmid as control plasmid in transformation to check if it gives colonies. To help you more, I need more information about the kit your using and any points were you vary from the original protocol.
    Best wishes
    Reply

    Posted by: AnonymousJuly 24, 2009, 4:56 PM

    Mark, I was using a Stratagene Kit. I used my input plasmid as the transformation control, and that dŒs give colonies. I gave up on the kit and tried your method. I get a few colonies, but now am not sure if the plasmid was mutated properly. It's a long and complicated discussion! I would type it if you have time to read it! :)
    Reply

    Posted by: AnonymousOctober 6, 2009, 11:59 AM

    Hi Leah, you can check your succesfull mutation by restriction digestion if you inserted or deleted the appropriate restriction sites with your mutagenic primers. If you are not able to introduce the sites together with your desired mutation you could first introduce a restriction site and then delete it by introducing your desired mutation. The introduction of a restriction site could then also serve as a control if the method is working. This would be the cheapest way. The easiest way would be to introduce your mutation without restriction marker and verify your successful mutation by sequencing of a number of colonies. When you choose this approach you could enhance the DpnI digestion to reduce background. However, you should always verify your mutation by sequencing before moving on with your experiments.
    Feel free to type your problems up, I would be happy if I could help you. Best wishes
    Reply

    Posted by: AnonymousOctober 8, 2009, 3:40 PM

    Unfortunately, I can't insert a restriction site because the area I would like to mutagenize is in the replication region, in the antisense RNA region. I went ahead and tried again with different primers and over and over, I get no colonies after transformation. I use about 60ng DNA, I have varied the annealing temp, tried longer extension times. Maybe I'm not using enough dNTPs? I'm pretty terrible at some of the math regarding concentration. :) I have a 40mM stock that I've been taking .5uL and putting it in a 50uL reaction. Should I be using 1uL? I'm also wondering if maybe the added mutations cause so much of a difference in the RNA that it can't function and thus, cannot be established in a cell? I'm getting minimal help from my lab, so any insight would be very much appreciated.
    Reply

    Posted by: AnonymousNovember 6, 2009, 2:59 PM

    Hi Leah, do I understand you right, your region of intrest lies in the origin of replication of the plasmid? If your region of intrest is not essential for plasmid maintainence you can try to mutate it and create a restriction site as a positive control. This one you can then mutate to your desired sequence, then using the absence of the restriction site as assay for succesful mutation. If your region of intrest is in the origin of replication and dŒs not allow any big changes you could verify your mutation by sequencing. Regarding the dNTPs, polymerases usually require a concentration of ²00µM or lower per dNTP. So just take 1µL of a 10mM dNTP mix for a 50µL reaction. Do you get a product on the gel after the reaction, check 5µL on a gel, you should see a distinct band. You may also try using electroporation for transformation, or try a different polymerase (proofreading, non strand displacing and producing blunt ends). Hope this helps you. Good luck.
    Reply

    Posted by: AnonymousNovember 11, 2009, 1:04 PM

    Hello,
    I was wondering if you need to do ligation with this protocol.
    Also, if the primers are complementary, is there much likelihood of producing primer dimers, and how can we overcome that?

    Thanks for you help
    Reply

    Posted by: Jyoti D.March 16, 2010, 12:01 AM

    Hi,
    ligation is not needed in this protocol. Normally no problems are observed with primer dimers if you stick to the protocol (ca ²5-45 bp primers with at least 8 perfectly matching bases on either side of the mismatched region). If you have problems, you can try to make the primers shorter. By the way, purification of primers (HPLC or PAGE) will also enhance the efficiency and accuracy of the reaction.

    Have fun
    Reply

    Posted by: AnonymousMarch 16, 2010, 5:53 PM

    i ordered the finnzyme,s (phusion) kit for site directed mutagenesis. and in that kit the primer designing was different. i have primers which are designed back to back and they are 5' phosphorylated(for lagation purpose). kindly guide me if i can use these primers with this protocol. coz unfortunately i didn get that kit. i have to complete this part of my work in a week. i m relly worried.
    Reply

    Posted by: AnonymousJune 2, 2010, 2:23 PM

    those primers are not overlapping and one of them have mutation (single). in taht kit they used hot start DNA polymerase. i have used the pfu polymerase. but got ambigous result. there were ² bands in pcr product. one of 3.5kb(size of vector(ptz) +my gene) and a 3 kb band. i wonder Y pcr produced ² bands :(
    Reply

    Posted by: AnonymousJune 2, 2010, 2:31 PM

    Hi Aliya, I would assume that you can exchange the phusion polymerase for the standard pfu (and reverse)as they both have the same basic functions (generating blunt ends, no strand displacing activity, proofreading). Also the orientation and design of the primers can vary a lot between different mutagenesis protocols available, so there are definetely many ways to mutate a plasmid. In your case I would say it's ok to use pfu instead of phusion as long as you use the correct cycling conditions. If you get two bands, you could either play around with the cycling conditions and/or primer design or you could just simply extract the band of correct size and use it for the ligation. Hotstart polymerases have several advantages (mainly higher specificty) but they are not necessarily needed. If you still have problems, you may also order new primers and stick with the protocol above. Hope this will help you.
    Have fun
    Mark
    Reply

    Posted by: AnonymousJune 7, 2010, 4:48 PM

    Hi Mark, i have recently ordered new primers (overlapping primers having mutation in middle of the sequence) but i i can,t add a restriction site in them coz mutation is in middle of my gene. and in ur protocol u didn mentioned about the size i mean the plasmid plus insert which u r using. my plasmid is of ².8 and my gene is about .5kb. would this procedure work for this size of DNA. and how the nick protroduced in the strands would be sealed in the end. and another question is in fermentas prescribed manual for pcr wd pfu polymerase it is mention that the extension time should be ²min/kb. which makes almost 1² min for my DNA. what should i do now.
    thanx for ur guidence
    Reply

    Posted by: AnonymousJune 15, 2010, 1:55 AM

    Hi Aliya
    If your desired mutation is in the middle of the gene and you dont want to insert "silent" mutations you can check by Sanger sequencing of a few clones. Mostly all of them carry the correct mutation. The size of your plasmid is totally fine for the protocol. For thermocycling you should definetely stick to the protocol of the supplier, if pfu is too slow for you just take phusion. Also I'm sure that not all 18 cylces we recommend in the protocol are needed. But generally the cycling times are quite long in this protocol, this is normal. The nick in the plasmid is repaired upon transformation in the bacteria.
    Have fun
    Reply

    Posted by: AnonymousJune 15, 2010, 4:06 AM

    Hi Mark
    thanx a lot for ur guidence. i followed that protocol and i succeded to introduce my desired mutation in gene. i just did it in 1st attempt. i got positive results by sanger sequencing. thanx :)
    Reply

    Posted by: AnonymousAugust 2, 2010, 1:42 AM

    can u tell me the mechanism of this method. how many copies of mutated plasmid will be produced by this method from pcr of single unmutated plasmid. i am a bit confused how the primers aneal and synthesis more copies from newly synthesized strands in next cycles of pcr.
    Reply

    Posted by: AnonymousSeptember 29, 2010, 12:35 PM

    Hi Shazar,
    the plasmid amplification works by a non PCR based thermocycling reaction (no exponential amplification but a linear one, similar to sanger sequencing just without disrupting nucleotides). The amount of mutated plasmid will be increased by the amount of input plasmid with every cycle, assuming the reaction is 100% efficient. So if you use 50ng of template, after the first cycle there should be 50ng of mutated plasmid, after the second cycle 100, after the third 150ng and so on. The amplification only takes place on the original plasmid and not on the newly synthesized (to my best knowledge). The newly synthesized plasmid strands then form plasmids with nicks in each strand which lie at the 5 prime end of each of the primers. If you transform this nicked plasmid into E. coli the nicks get repaired and result in a normal plasmid. I think in the video there's a scheme of the amplification process. If you cant acces the video you can view the article which the viedo is based on (in german). It contains the same scheme. www.laborjournal.de/rubric/tricks/tricks/trick1²6.lasso
    All the best
    Mark
    Reply

    Posted by: AnonymousSeptember 29, 2010, 5:28 PM

    Hi,
    I have the problem of primer dimer with this protocol and i did not find any band after gel then i redesigned the primer which are partially overlaping and it works well in the first time and i got a good band
    but when i repeat it again it failed -----i used the same primers but changed only one amino acid in the same position
    i do not know what should I do ?
    any advise will be helpful to me
    Reply

    Posted by: AnonymousJuly 7, 2011, 10:43 PM

    Hello Friends..
    Well iam working on Site-directed mutagenesis of one gene coding for one enzyme,however i dont get desired mutant ,sequencing results in undesirable mutation beside desired mutation. I follow strategene quick change mutagenesis protocol. I use pET²8a vector.Could anyone please guide how to adress this issue..Thanks fr d help..
    Reply

    Posted by: AnonymousAugust 12, 2011, 5:14 AM

    Hi Shad,
    have you made sure that the undesired mutation was caused by the mutagenesis and was not present already before? If the undesired mutation appears in a region outside the one you are mutating, simple check some more colonies. As the mutagenesis is based on a linear amplificaation undesired mutations should not be propagated and therefore not present in most of the colonies. However if the mutation lies in the region of your primers, go and check their sequence once again (on the tube). During mutagenesis your primers are incorporated into the new plasmid, so if these are corrupted you will never get a good result. You could as well go for HPLC purified primers, which diminishes the risk of having a mixture of primers. Hope this helps you.
    Enjoy,
    Mark
    Reply

    Posted by: AnonymousAugust 12, 2011, 8:24 AM

    Thanks dear for your kind reply,
    i have ensured the mutations were not present prior amplification. Mutations were not in the primer as it is FPLC- HPLC purified. I get many deletion and insertion kind of muations. i even changed the Polymerase, from Pfu polymerse to Ex taq polymerase, still didnt work out.. Please help me out with a nice solution to this problem.. always thanks..
    Reply

    Posted by: AnonymousSeptember 6, 2011, 10:20 PM

    Ok, so the mutations seem to appear during mutagenesis and they are additional to the desired ones which you introduce with your primers, right? I'm not sure where the mutations lie relative to the primer but you must be aware that in sanger sequencing the read quality towards the end gets quite poor and might seem like a mutation. To ensure that what you are seeing is really an additional mutation, you could check the chromatogramm and also perform a second sequencing reaction from the other side to ensure you have high quality coverage off the putative mutation. But this only if the chromatogramm quality is poor. Otherwise, I would say, try a diffrent polymerase such as phusion hotstart. Maybe you could also go for a fresh aliquot of dNTPs. Sorry Shad for the delayed answer, but keep the comments coming if you've got problems. Hope this will help.
    Reply

    Posted by: AnonymousSeptember 18, 2011, 8:43 AM

    Hey Thanks Dear Mark for responding to my queries.!
    Yea you r right, i get final additional mutations in the gene sequencing result[Macrogen]. The undesirable mutations found through out the gene not at any specific position relative to primers, when i checked by aligning the wild & mutant gene using BiEdit program. I change the polymerase to Ex taq polymerase, i get the desired band of expected size, however confirming its sequence results in failure due to same additional undesirable mutations.
    I dont have much idea about Chromatogram and about sequencing from other side? As we sent our sequencing to Macrogen Gene sequencing Comp. Could you please tell me little more about it.
    Always Thanks. Your suggestions surely helps me. Hope to keep getting your valuable guidance.
    Reply

    Posted by: AnonymousSeptember 18, 2011, 11:10 PM

    Hi Shad, what I mean with sequencing from the other side is simply that you should make sure the sequencing reaction gives reliable results. So what we do when we check a plasmid is sequencing into the orf from both sides, 3 prime and 5 prime ends. You could as well use a primer which lies further into the gene but sequencing in the same direction. When you get your sequecing results there should always be file containing the unprocessed chromatogram for example .abi file. You can check by opening the file with finch.tv program. Once you have the correct files, use these as input for the alignement and compare to the reads you get before mutagenesis. Extaq I don't know but generraly you should only use enzymes with proofreading activity (pfu based enzymes or kod should work). Ok and as well there is no expected band size because the reaction amplifies the whole plasmid. Maybe you can get some help from your labmates or the sequencing company regarding the quality of reads. Good luck and keep me posted. We will get it solved :-)
    Reply

    Posted by: AnonymousSeptember 20, 2011, 3:55 PM

    I did this protocol for getting point mutants and i have been successful a couple of times. But recently i am noticing the sequence has multiple copies of mutated oligos. The primer sequence is concatenated and repeated several times in the mutated plasmid. The primers i used had a tm of 65 C and are ²8 bp long. What could be the problem ?
    Reply

    Posted by: AnonymousOctober 7, 2011, 10:18 AM

    Hi Sankar, I have not yet encountered the problem you describe. However I could imagine it is caused by pairing of the primers at their ends. You could check if the sequences could allow such a pairing and the maybe add some more bases to dimish dimerisation. You could also try raising the annealing temperature. Keep me posted. Mark
    Reply

    Posted by: AnonymousOctober 7, 2011, 11:55 AM

    Hi Sankar, I have not yet encountered the problem you describe. However I could imagine it is caused by pairing of the primers at their ends. You could check if the sequences could allow such a pairing and the maybe add some more bases to dimish dimerisation. You could also try raising the annealing temperature. Keep me posted. Mark
    Reply

    Posted by: AnonymousOctober 7, 2011, 11:55 AM

    The problem I believe is, while the polymerase is amplifying, the primer spontaneously separates allowing the polymerase to copy the primer region on to a daughter strand. Since the DNA is circular two primer regions are close by in the daughter strand. The reverse primer could anneal in this region and the polymerase could amplify the daughter strand instead, creating multiple primer regions on subsequent PCR cycles. In a normal point mutation reaction, the amplified daughter strands should not act as a template.

    To solve this, i ran the cycle separately for the two primers in two different tubes and mixed the samples before dpnI digestion. I had several colonies and one in 10 were positive for the mutation. And sequencing gave no concatenated primer region in any of the positive clones.
    Reply

    Posted by: AnonymousNovember 4, 2011, 5:33 AM

    Hey mutagenesis fans, if you have problems mutating large plasmids you can give this modified protocol a try. In a nutshell, the protocol works for well for large plasmids, makes use of KOD polymerase and only 6 amplification cycles are needed: http://www.zju.edu.cn/jzus/openiptxt.php?doi=10.1631/jzus.B1100180 (download quite slow but eventually works). Good luck and happy pipetting
    Reply

    Posted by: AnonymousNovember 2, 2011, 2:17 PM

    I have amplified ss DNA library of 60 mer size by assymetric PCR method using gradient primer(F.Primer 100uM R.prmer-10uM). i could able to amplify ss DNA libray ,however fail to isolate amplified DNA band from the Gel slab by Crush & Soak protocol.
    Thanks.

    Reply

    Posted by: AnonymousJuly 16, 2012, 9:44 PM

    Hi all,
    I could't figure out how to us NEB tool to design primer bearing a new restriction site, which can be used for clones screening. could anyone share any tips on how to conveniently do so?
    Many thanks!
    Reply

    Posted by: Oleksiy K.March 20, 2013, 1:38 AM

    Dear Oleksiy K.

    My name is Ana Egana and I am the Technical Support Manager at New England Biolabs. I would like to invite you to contact us directly at info@neb.com with your questions. We will be happy to review the tool with you and provide you guidelines on how to use it for your intended purpose. We look forward to hearing from you.

    Sincerely,

    Ana
    Reply

    Posted by: Ana E.March 26, 2013, 10:10 AM

    Your Figure 1 seems to be broken.
    Reply

    Posted by: August P.March 21, 2014, 12:11 AM

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