Hi!
I am using the "Roboklon DNA core kit" to perform the SELEX. This company proposed the amplifications steps with an emulsion PCR. But I've compared with normal PCR and RT-PCR, and this emulsion seems to be not so good. Someone had some problems with this kit and this emulsion PCR?
After each round I have to clean the PCR product with a column (provided in the kit) and I am losing about 80% of my product after column. Is it normal?
And checking each round in the agarose gel, is possible to see 2 bands. What is happening?
In the RT-PCR after 10 rounds I did not see a increasing in the melting curve (stopped around 85 degrees). Am I doing something wrong?
-> In this kit, the library is compound by ssDNA (40 bp) flanked by 18 bp (for the primers).
Thank you!
Hi Sabrina,
We recently bought the Xelex kit also, we are having problems obtaining 1ug of library for the next round, we get there after two ePCRs (15 cycles each) with 100ul of water phase with the eurx mastermix taq. How did you calculated the amount of library you were losing after purification? Did you find the reason behind the two aands in your gel. Also, could you help me with the amount of beads you use? My beads are 1uM and haave a binding capacity of 15pmol/ul and the manual is quite confusing about this part... thanks
Hola Katherin,
Puedo contestarte en español?
Bien, yo intenté muchas veces hacer las selecciones con el kit de Roboklon.. Pero después de muchos meses vimos que no funciona bien. Entonces pedí nuevos primers, nueva librería y hizo las soluciones en mi labo. Y creo que me funciona bien así.
Cuanto a la eficiencia de la purificación del producto de PCR después de cada round, descubrí que no se puede centrifugar a una velocidad muy alta, pues como son fragmentos cortos ellos bajan de la columna. Entonces utilizo a 9.000 RPM por 30 segundos (con el kit de purificación de Qiagen).
Sobre las dos bandas no sabemos muy bien lo que es. Imagino que los primers y el dNTP se agota con muchos ciclos y así se forman bandas inespecificas... Una solución para esto fue hacer el primer round en RT-PCR y así es posible ver la evolución de los ciclos y parar hasta uno determinado nivel.
Tu estas seleccionando aptamers para proteínas? Lo que ago es utilizar 125ul de beads en ~ 100ug de proteína (con His tag), incubar por 20 min, lavar y después resuspender en 333ul SELEX 1x. Para las selecciones utilizo estas beads con un volumen decreciente hasta el ultimo round. Y a cada round yo subo el número de lavados.
No sé si te contesté todas las preguntas. Imagino que tienes un montón... Si quieres puedes enviarme un correo y te explico mejor, [email protected]
Suerte en tus experimentos!
Sabrina
Hi I am thinking of ordering this kit for selection of my own aptamers. Did you manage to overcome the issues to produce good aptamers?
Hello Catherine,
I did not like this kit. Actually I am using other library and is working better.
Hi Sabrina,
emulsion PCR is very different from normal PCR. The yield cannot be high if the emulsion stays intact during PCR and you have little input, so very few droplets are filled with a template. This is why you may need to do 2 consecutive ePCRs. However, if you do the normal, biased PCR for many selection rounds you will quickly run into artifacts, especially if you overcycle.
My Spanish isn't too good, but you seem to be doing RNA SELEX involving RT-PCR. We have discovered that most reverse transcriptases (RT) will not work in emulsion. It RT-PCR means "real-time PCR" instead, the emulsion is opaque and not compatible with fluorescent dyes as necessary in "real-time PCR".
The greatest problem with SELEX is the target. For a multitude of targets no normal DNA will be able to bind, because of electrostatic repulsion. Positively charged targets are generally better, but may in some cases be too sticky to find specific binders, either. Sometimes it may be better to use RNA aptamer libraries instead. Unfortunately, the general target problem cannot be overcome by any kit. With SELEX you'll always have a higher risk of failure compared to protein-based methods like phage display. Yet, proteins and peptides have other issues again.
Regards,
Jörn
Hai, is anyone using the SELEX kit from InnovoGene Bioscience? Is there any problem when using this kit?
Hi Woon,
InnovoGENE Biosciences is a new company spun off from McMaster University and Dr. Yingfu Li's lab who is a leader in nucleic acid research and aptamers. If you have any questions, Dr. Kha Tram, the CEO, would be only happy to advise on your questions and assist you with your project. He can be reached through the company website. The Li Lab at McMaster University is currently using InnovoGENE's SELEX libraries.
Disclosure: I am an affiliated clinician/researcher with the Li Lab and I am also affiliated with InnovoGENE.
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