Determine+titer+of+library+and+next+step+10-12-12

I have some plates which will allow me to determine the titer of the library. I will count them soon.

I need to find out what to do with the library once the titer is determined though. The next part of the In-Fusion SMARTer Directional cDNA Library Construction Kit manual talks about doing the amplification of plasmid libraries part. I am quite sure that when I was at the step with the mouse tumor cDNA library that I did not do such an amplification (it would require way too many LB plates). I either just stored the in-fusion product without doing the transformation or I just did a miniprep of the transformed bacteria. From my previous lab notebook notes from around 5-3-11, 4-13-11, etc. I seem to have just transformed the amount of DNA I needed to determine transformation efficiencies. I will determine the current titer of my library, send for sequencing to determine the complexity, and then most likely do a miniprep of the remaining transformation solution or just dilute these cells to start producing protein directly.

Note that when I transformed the mouse tumor cDNA library, DH10B cells were used which are the same types of cells that were used this time.

Is there enough ds cDNA leftover to repeat the In-fusion reaction if necessary? No I don't think there is. There is some 1st strand DNA reaction though so that could be used.

The number of cells on the negative control plates have just about as many colonies as the cells on the library plates. Therefore, it is probably best to remake the library. Next time I think I will do the transformation using my protocol (SOC instead of LB) rather than the manual protocol. - I can still try to estimate the total number of colonies in each transformation tube though. "C:\kurt\storage\CIM Research Folder\DR\2012\10-17-12\hum cDNA\colony counts 10-17-12.xlsx"

I only have a total of 15,800 colonies in the tumor transformation solutions.

Note that the negative control in this experiment was just linearized vector with no insert.

These samples were miniprepped and stored at -20 C

Items: AT, BT, CT, AC, BC, and CC at 10-17-12 MM in Human tumor cDNA 8-3-12 -20 C