October 31st to November 20th

In order to obtain bands for the 5' end we are ordering the pfx taq that is recommended in the generacer kit and I will be running a control on this as soon as we receive it. I repeated the cloning reaction on the 3' ends obtained in PCR, but was unable to do the control because we don't have kanamycin. We were using ampicillin screening on the colonies. I cloned the PCR products I did have and a few colonies grew on each plate. To purify the colonies I performed a mini prep, using two different colonies from each plate. I nanodropped the results of the mini prep and do have ample DNA. Using the purified plasmids, I am now performing PCR to excise the DNA sequences in order to use them in sequencing.
Hopefully over the next month I will be able to perform sequencing on the 3' ends and overcome the challenges on the 5' ends. Unfortunately, oligo capping doesn't always work but I am still hopeful that we can find a way to get results with this procedure.

October 10th to October 30th

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.