1. PCR AMPLIFICATION CYCLE AND PRIMER CONCENTRATION OPTIMIZATION EXPERIMENTS
>PCR optimization conditions: our reagent and Qiagen reagents
Since PCR products, that were obtained using combination of our own reagents (Taq.pol from New England Biolabs, MgCl2 from NCB and so on), were faint on gel I decided to use Qiagen PCR amplification kit, which I found in the freezer. I run two reactions at the same. As the result bands for PCR products from Qiagen were more visible and stronger.
Conclusion: use Qiagen reagent kit for further PCR amplifications
Problem: still having multiple bands and smears
Primer optimization: primer set 20uM, Primer set 0.5uM
According to Qiagen kit the primer concentration for PCR amplification should be 0.5uM. So I did PCR amplification for primer concentration that we have been using and primer concentration according to Qiagen protocol. PCR amplification worked only with 20uM primer concentration.
Conclusion: keep using primers that you have been using
Problem: still having multiple bands
Preparation for SELEX 9; PCR optimization: cycles 10, 12, 15 and 20
To resolve problem with multiple banding I decided to use two different cycles 15 and 20. Then I further gel purified band from 15 cycle run.
Conclusion: In 15 cycle PCR amplification program there is less by product comparing to 20 cycle PCR amplification program
Problem: Still having multiple bands
After gel purification of SELEX 8 product, I have set up another PCR amplification, but this time with 10,12 and 15 cycles. According to the image cycles 10 and 12 gave less by products whereas cycle 15 didn’t. I have used two set of DNA template for cycle 15: first from SELEX product 8 and second from SELEX product 8 that was gel extracted. This time gel extraction didn’t gave us anything exciting.
Conlclusion: Gel extraction method didn’t work this time. Use cycle 12 for further PCR amplifications.
2. SELEX 7-12 RESULTS
CONCLUSION:
12 cycles of SELEX procedure were completed successfully. The next step is analyze their affinity to CEA using DOT-Blot Assay. Also start to clone SELEX 11 and 12 products into E.coli.
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