This has been a busy few weeks for me. I began by cloning the extracted bands for the 3' ends of each gene using a TOPO TA cloning kit and TOP10 chemically competent cells. Nothing grew on these plates so I am going to run a positive control and replate the excised bands at the end of this week hopefully.
I used the newly prepared cDNA to run PCR again on the 5' ends of each gene but still no bands were visible. There was cDNA produced in the capping and cDNA strand synthesis so I am going to try and optimize the PCR reaction some more. After speaking with Jessica, we decided to go ahead and order the Platinum Pfx DNA polymerase that the kit calls for and also run a positive control simultaneously with the generacer reaction.
I am still trying to get the reactions to work and have a few new things that I will be working on this week and next. I will let you know how it all goes and if I have any more success.
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