Week 11: Pufall Lab, University of Iowa

August 7th, 2014

John Christiansen ’15, Dimensions Fellow in Research

This week has been spent trying to get the deletion check primers to work. These PCR primers would be used to amplify a region of transfected NALM6 genomic DNA to determine if the CRISPR vectors were effective in cutting out their target. However, previous attempts to validate the effectiveness of these primers using wild type NAML6 DNA have not gone well. I later learned that the primers were misordered. This week we received the proper primers and I began to work with them.

So far, I have performed the PCR with these primers three times. The first time (Friday, August 1st) I found that none of my samples, even the positive control, had any product bands when analyzed with gel electrophoresis, though the 100 bp ladders still showed up clearly. This suggested that something had been wrong in the reaction, and on closer examination it was found that I had added too much dNTP. PCR can be finicky and this might be why nothing was amplified. On Monday (August 4th) I tried again. This time the correct dNTP concentration was used, but the only product bands seen were two very faint bands, each in no template control lanes. Oddly enough they were the right size for the primers used. The procedure was performed correctly, so the fact that I saw so few bands suggested that my reactants were somehow inept and the fact that I saw bands in the no template control suggested that I had DNA contamination. The solution to both problems was to replace each reagent and try again. I also reduced the amount of ladder I loaded in with my samples after being informed that the imaging machine we use to see the bands generates an image based on relative intensity.

The third time I tried PCR with the correct primers (August 5th) I used new reagents and only loaded 3 uL of the ladder with the samples. I finally got some positive results. Out of the five primer sets tested, two had worked very well. One had replicated the desired product well but had faintly replaced a few other bands, and another produced appropriately sized but faint product bands. Only one of the five sets failed to produce a product band. These results were not perfect, but it was a relief to finally see positive results. Later today (August 6th) I’ll be redoing the three sets that didn’t work as well with some different conditions to see if I can optimize them.

Of the five primer sets, two were for my work with BCL6 and the other three were for different gene that the lab technician Mimi had been working on. My two primers worked out well, and I am ready to move on with deletion screening as soon as Mimi has the time to show me how that procedure works; hopefully I’ll be able to pick it up from watching her work with her primers. I also have assembled the samples I needed to test the dexamethasone induced BCL6 regulation in SUP-B15 cells. I have previously do the same with NALM6 cells, but a few complication have previously delayed my work on SUP-B15. I look forward to retesting the qPCR skills I have developed this summer.

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