Jon, Thank you for your detailed answer. I will be using next-gen sequencing for the final step. I like your suggestion on barcoding the eluates at different stages as the aptamers emerge. 

Also, I came across this fast protocol called "RAPID-SELEX" which I'm yet to evaluate:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082667

 I am not sure about the quality of the TriLink library, also I dont know what is the length of the random region. Most common ones are either 40 or 50 nt long. I would argue that ou have a much better chance of identifying a high affinity aptamer to a given target with a larger random region library and a well characterized library. I have generated an N70 library and verified it randomness carefully with next gen sequencing. 

I am an author on the RAPID-SELEX protocol paper.

Jon, there is actually a high-throughput assay one can use to get Kd values for millons of RNA aptamers simultaneously. You may want to check HiTS-RAP paper in Nature Methods. For DNA aptamers one can use a modified version of HiTS-FLIP method by the Burge Lab, published in Nature Biotechnology.

Abdullah, great to have you on this discussion. I gather that you custom-made the N70 library through GenScript, and then added the T7 promoter sequence. Does your lab have a step-by-step protocol for RAPID-SELEX? The PLOS paper is pretty detailed, but I thought you might have updates on it. 

Yes, I made the custom N70 library. I would not recommend you doing that, cause it took me ~6 months to make it. I have optimized every single step to prevent introduction of any bias into the library. It took 1 L ( 1 liter) PCR to amplify the library, which I did it the old fashion way using waterbaths and manual cycling on tubes, 100 ml PCR per day. Also, you can not directly use the oligo synthesized by the company to make your library in PCR. Those libraries contain many oligos that are not PCR amplifiable as they arrive. After PCR I did 88 ml T7 transcription. As I said before, I checked every step to verify that we did not introduce any bias into the library (other than the slight bias present in the original synthesized library). There are not that many N70 libraries out there, and even the shorter ones are not as carefully made.

You might also want to check on the Scientific Reports paper that was recently published. We have done the RAPID-SELEX in a slightly different format: 1st round 1 cycle (non-RAPID), 2nd round 2 cycles (RAPID), 3rd round 3 cycles, and 4th round 4 cycle. So total of 10 cycles of selection, in 4 round (=4 amplification cycles). Much faster than original RAPID protocol.

I dont think what we have (the step-by-step protocol) will be any use to you, since it is designed/optimized for our microcolumn devices (MEDUSA and etc..). 

Hi. I am new to this field and I would like to learn more about aptamers and and work on their diagnostic applications. Would very much appreciate if anyone could give me inputs on the things to take care while starting some pilot work in this field. Initially I am looking at procuring some well established aptamers for targets like pathogens and looking at how they fare in detection of the said

pathogen in clinical samples. I am a medical microbiologist with not much experience in cloning or sequencing. Would be grateful for any inputs on this.