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

2015-01-13

azim58 - 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

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