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igenetic

Москва

07.10.2019 17:19

URL #1

Коллеги, может быть вы сталкивались с похожей ситуацией.

Амплифицирую плазмиду размером 4,5 кб с полимеразой Phusion HotStart Fermentas.

Реакцию ставлю с двух фосфорилированных праймеров. Один прямой имеет с 3'-конца копмлементарные матрице 20 нуклеотидов и 20 нуклеотидов на 5'-конце некомплементарные. Обратный праймер полностью комплементарен части матрицы с tac-промотором, таким образом, что на его 5'-конце находится ТА-богатая область -10.

Так вот. Реакция проходит, и при последующем лигировании плазмида зашивается в кольцо, НО. Почти во всех клонах содержится делеция со стороны R-праймера, где область -10. Как правило отсутствуют все 6 букв, иногда меньше. Этот праймер перезаказывала новый и переставляла реакции с разными температурами отжига. Загадка в том (повторюсь), что делеция в итоговой плазмиде только со стороны обратного праймера, в области -10.

Может быть у вас есть какие-то мыли и рекомендации, как избежать такую делецию?

Сообщение было отредактировано igenetic - 07.10.2019 17:26

guest: petr
IP-штамп: frpzMrmKUuRMg
гость

14.10.2019 20:58

URL #2

Посмотреть бы на обратный праймер...
Ну первое что приходит в голову, образование вторичной структуры на конце праймера или амплифицированной плазмиды. Если есть возможность, то я бы удлинил обратный праймер с 5`конца чтобы сделать этот конец более тугоплавким

Herr Opsis

15.10.2019 07:15

URL #3

https://link.springer.com/protocol/10.1385%...259-273-2%3A051

Herr Opsis

15.10.2019 07:16

URL #4

(guest: petr @ 14.10.2019 21:58)

вроде ни прямого ни обратного девушка не выставила

Herr Opsis

15.10.2019 07:26

URL #5

http://arep.med.harvard.edu/labgc/estep/lo...R_protocol.html

Herr Opsis

15.10.2019 07:44

URL #6

Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration.

Herr Opsis

15.10.2019 08:09

URL #7

берите плимераз микс нонпруфрид и пруфрид типа клентака и пфу в отношении 10 к 1
праймеры около 40 нт
тяните на 70 дегризов

Herr Opsis

15.10.2019 08:30

URL #8

у меня таже проблема задолбали эти уже пруфрид полимеразы

genseq
Постоянный участник

15.10.2019 10:01

URL #9

Если делетируется промотор, то причиной может быть штамм-реципиент. При отсутствии в нём репрессора усиленная транскрипция любого гена будет губительной. В результате при трансформации идёт селекция вариантов с повреждённым промотором. И не стоит полагаться на паспортные характеристики штамма-реципиента. Плазмида с репрессорным геном может теряться.

ship
Постоянный участник
Moscow

15.10.2019 15:38

URL #10

конечную цель манипуляций опишите. то есть задача какая? вставить в плазмиду 20 нуклеотидов?
если да, то делайте по quikchange протоколу с двумя комплементарными праймерами

igenetic

Москва

17.10.2019 15:09

URL #11

(ship @ 15.10.2019 16:38)

Задача: просто замена 24 нуклеотидов

igenetic

Москва

17.10.2019 15:11

URL #12

(genseq @ 15.10.2019 11:01)

Спасибо за мысль! Но в данном случае это исключено, потому что контрольные варианты прекрасно трансформируются и поддерживаются.

ship
Постоянный участник
Moscow

17.10.2019 15:17

URL #13

(igenetic @ 17.10.2019 13:09)

Задача: просто замена 24 нуклеотидов

Делайте по Qiukchange без вариантов. правда праймеры будут длинные. если у вас мало денег или плохо варят праймеры - то могут быть проблемы.

кроме того, чтото мутировать в экспресионной плазмиде - идея не очень хорошая. Ибо после ПЦР у вас нет гарантии того, что гдето не случилась ошибка.

Поэтому я бы вырезал целевой кусок из плазмиды по уникальным рестриктазам ,засунул бы его в любой стандартный вектор, и уже там вставлял 24 нуклеотиды, секвенировал, и потом обратно в конечную плазмиду.

igenetic

Москва

17.10.2019 15:25

URL #14

(guest: petr @ 14.10.2019 21:58)

R-праймер 5' - 3' cattatacgagccgatgattaattg

В данном случае этот праймер может быть таким и только таким. Увы, удлинить не могу

igenetic

Москва

17.10.2019 15:31

URL #15

(ship @ 17.10.2019 16:17)

Вариант с такой сложной заменой через еще один вектор -- не подходит. Потому что вся задача и состоит в том, чтобы быстро менять эти 24нт, заказывая каждый раз только один F-праймер с различными 24нт.

Про квикчендж сейчас почитаю, спасибо!

guest: petr
IP-штамп: frvKCqje1sDbE
гость

18.10.2019 06:06

URL #16

Если не хотите через второй вектор, то или квыкчейндж, или смотрите по бокам от потенциального места замены уникальные рестриктазы и делаете мутагенез ПЦРом в 2 этапа. В результате ПЦРов у Вас будет "вставка" по этим рестриктазам с нужной Вам последовательностью.

Вам надо сделать 2 ПЦр продукта с перекрывающейся областью (конец первого - перекрытие с началом второго этак на 20-25 нуклеотидов).

Соответственно у Вас 4 праймера:
FW для правого куска: 5`-6 букв + сайт рестрикции + комплементарная часть-3`
Rev для правого куска: 5`-20-25по перекрытие + комплементарная часть-3`

FW для левого куска: 5`-20-25по перекрытие (комплиментарно перекрытию Rev от правого праймера) + комплиментарная часть-3`
Rev для левого куска: 5`-6 букв + сайт рестрикции + комплементарная часть-3`

ПЦР:
1. ПЦРите правый и левый куски отдельно (если с плазмиды, то не надо больще 15 циклов делать)
2. смешиваете по 1 мкл из предыдущих ПЦР реакций и ПЦРите с внешними праймерами (тоже самое, 15 циклов - более, чем достаточно).

Далее:
Режете/клонируете.

Удачи Вам!

igenetic

Москва

18.10.2019 13:28

URL #17

(guest: petr @ 18.10.2019 07:06)

Спасибо!

Asterix
Постоянный участник

18.10.2019 23:54

URL #18

Однажды мы имели такую проблему , ничего не помогало. Ровно один нуклеотид на конце вставки регулярно исчезал при клонировании ну и рамка считывания сдвигалась.

Решили проблему перекодировав участок вокруг нуклеотида.

Попробуйте, измените последовательность этих шести нуклеотидов так чтоб оно ни на что существенное не влияло. А вдруг сработает еще раз.

igenetic

Москва

21.10.2019 13:29

URL #19

(Asterix @ 19.10.2019 00:54)

Тоже классная идея, возьму на заметку! Спасибо!

Поблагодарили (1): Asterix

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Постоянный участник

04.11.2021 10:04

URL #20

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Постоянный участник

18.11.2021 12:21

URL #21

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24.11.2021 11:48

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If you want to know more about it then you can visit our website Disneyplus.com/begin and take advantage of it. The Amazon My TV Code program requires the customer to have an Amazon account and a streaming device or TV. Amazon Prime Video is available on almost all streaming devices like Roku, Amazon Fire TV, Chromecast. All you have to do is go to Amazon.com/mytv and activate amazon.com/mytv with the help of Amazon's activation code. Webroot Antivirus is the fastest and easiest software to download, install and run.Webroot.com/safe In addition, it automatically updates without slowing down your system. Because of which you always get the most protection. So we recommend you to download Webroot Antivirus. Webroot Antivirus detects online threats and blocks them quickly.The need for a reliable antivirus like Norton Enrollment is increasing, you can download it by visiting the website Norton.com/setup Not only companies as a measure of data security, but everyone is now relying on antivirus programs that can be found on the norton.com/setup with product key page. If you have any problem you can visit our website norton.com/setup and get help.Mcafee.com activate- Find the easy way to mcafee.com/activate download, install and activate McAfee Antivirus, type the 25-digit product key at mcafee.comactivate. Protect your PC from virus, spam, spyware, and malware. McAfee provides the latest antivirus software, spyware removal and internet security for your home or home office.Present mcafee thing with mcafee.com/activate no issue. We give best foundation advantage.

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IP-штамп: frNpECYifupYs
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24.11.2021 12:15

URL #35

Microsoft only keeps a record of Office setup enter product key if you purchased from the Microsoft online store. You can find out if you purchased from Microsoft in your Microsoft account Order history.office it the pile of office www.office.com/setup gadgets to make your working smooth and effective.visit: to downloaded in your PC with the smart help.Endeavor which is amazingly office.com/setup easy to present, download and recoup.Norton Internet Security is a widely used security. If you want to get Norton Internet Security then you can go to norton.com/setup.office it the pile of office Office setup gadgets to make your working smooth and effective.visit: to downloaded in your PC with the smart help.Endeavor which is amazingly office.com/setup easy to present, download and recoup.The need for a reliable antivirus like Norton Enrollment is increasing, you can download it by visiting the website Norton setup product key Not only companies as a measure of data security, but everyone is now relying on antivirus programs that can be found on the norton.com/setup with product key page. If you have any problem you can visit our website norton.com/setup and get help.Norton Internet Security is a widely used security.

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IP-штамп: frNpECYifupYs
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24.11.2021 12:29

URL #36

webroot Mind blowing, lightweight, fused protection for PC, Mac and Android, cloud-based Webroot Internet Security Complete with antivirus guarantees singular information by thwarting the latest malware, phishing, and advanced ambushes. Visit to Webroot download present webroot antivirus in your pc. Download and introduce Webroot Software and Webroot Secure Anywhere AntiVirus which influence your PC and workstation.Here are the features www.webroot.com/safe of webroot antivirus : Secure up to PCs or Macs, Protect yourself from identity theft and No time-consuming updates or slow scans. For Taking benifits get installed Webroot.com/safe it in your PCs.Canon.com/ijsetup mg3650 – Canon offers a few finishes in with no reservations ones in this section and the PIXMA MG3550 is Canon's most recent advertising.The Canon Pixma TS3300 All-in-One Printer, easily replaceable 2-cartridge FINE Hybrid Ink System, enhanced wireless setup, a rear paper tray for quick and easy reloading of plain or photo paper and flatbed scanner from your basic canon ts3300 printer more. In addition, ij.start.canon ts3300 can print from smartphones or tablets, laptops, and computers with built-in Wi-Fi using AirPrint, Mopria Print Service, and the Canon PRINT app.Before you begin the setup process, unpack the printer. Check if the package contains all the components needed for the setup. If you wish to connect your ij.start.canon printer to the computer with a USB cable, then get one from the retail store.

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IP-штамп: frNpECYifupYs
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24.11.2021 12:43

URL #37

Sign in to your Hulu account. A Hulu account gives you access to an amazing selection of movies, TV shows, music and more from the www.hulu.com/activate Channel Store. With thousands of available channels activate my hulu account visit here for more details.Canon gives you access to the canon printer drivers suitable for your printer. Visit canon official website to download the canon setup mg2550 & Software then install and set up Canon Drivers in your PC or laptop. Here we go through certain quick and easy step-by-step instructions for Canon/ij setup mg2550 Setup which includes the printer unboxing and configuration guidance.Canon MG2522 is an all-in-one color inkjet printer. It can scan and copy your documents. This printer is supported on both - operating systems like Windows and Mac. This blog is to tell you how to connect the Canon.com ijsetup mg2550s printer to wifi.Mcafee antivirus acts as a shield of the device. It protects against viruses, malware, online threats etc. You can download, install and activate mcafee.com/activate with activation code.

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IP-штамп: frNpECYifupYs
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24.11.2021 13:06

URL #38

To install, activate and redeem the Mcafee card on your PC, you must have a product key or mcafee.com/activate activation code.Once you successfully purchase Ms. Office Equipment, you will get an Office.com/setup ,in which you must enter the key.Mcafee antivirus acts as a shield of the device. It protects against viruses, malware, online threats etc. You can download, install and activate mcafee.com/activate with activation code. mcafee antivirus provides protection from viruses, hackers, spyware and more harmful websites. How do I get my Download IJ Scan Utility Windows 10 Scanner Software? Download ij scan utility is an application that allows you to scan photos, documents, etc easily. You can complete from scanning to saving at one time by simply clicking the corresponding icon in the IJ Scan Utility main screen.Sign in to your espn account. People's media needs can be met through the Amazon Prime platform. Amazon Prime is a subscription service, which can be subscribed by going to the link Amazon.com/mytv.The Amazon My TV Code program requires the customer to have an Amazon account and a streaming device or TV. Amazon Prime Video is available on almost all streaming devices like Roku, Amazon Fire TV, Chromecast. All you have to do is go to Amazon.com/mytv and activate Amazon.com/mytv with the help of Amazon's activation code. You can visit our website amazon.com/mytv for details of the entire process.

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IP-штамп: frNpECYifupYs
гость

24.11.2021 13:27

URL #39

To setup and download the Hp priter driver software, visit our website 123.hp.com/setup.The 123 hp setup HP printer makes printing hassle-free and easy.The Amazon Prime Activation Code is a six digit alphanumeric code that can be found on your TV screen when you click on “Register” on Amazon.com/mytv .Amazon prime paid for online streaming services used at Amazon.com/mytvGives users access to a variety of services such as instant delivery.Search for “Amazon” to find the amazon.com/mytv app.Get more information.Mcafee antivirus acts as a shield of the device. It protects against viruses, malware, online threats etc. You can download, install and activate mcafee.com/activate with activation code.Office it the heap of office devices to make your office.com/setup working smooth and effective.visit: to downloaded in your PC with the snappy help.office setup is the best programming which is Office.com/setup comprehensively used in globe .It is an astonishing affiliation that causes you discharge your best considerations, complete things, and remain related on the go.for more nuances visit: office.com setup today.office it the pile of office gadgets to make your Office.com/setup working smooth and effective.visit: to downloaded in your PC with the smart help.

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