pentadecamer+library+post+in-fusion+4-19-13

4-19-13

Finished ethanol precipitation after the in-fusion.

Ran 1 uL of each sample out onto a gel.

see "F:\kurt\storage\CIM Research Folder\DR\2013\4-19-13\electroeluted library\gel of electroeluted library 4-19-13.svg" This is a gel of the electroeluted library after ethanol precipitation, an in-fusion reaction, and another ethanol precipitation.

Estimated percent recovery from original cDNA synthesis amount to the cDNA now after an electroelution, ethanol precipitation, in-fusion, and another ethanol precipitation. 132*"ng"/(2133*"ng")*100.0 = 6.19% see 4-10-13 experiment for the gel of the original cDNA which was used to determine the 2133 ng amount.

1 = 4T1 cDNA created from random pentadecamers (13.2 ng/uL) 2 = Positive control eluted (2.7 kb PUC19 and 2 kb control insert) (0.17 ng/uL) 3 = Positive control (2.7 kb PUC19 and 2 kb control insert) (5.97 ng/uL) 4 = Negative Control (2.7 kb pSMART2IFD) (0.46 ng/uL)

-Electroporation Now I need to decide how I want to do the electroporation reaction.

How do they do it in the SMARTer In-Fusion directional manual? They just transform their full 10 uL In-fusion reaction without quantitating anything.

How do I usually do it? I use 10 pg to 100 ng. Let's say I use 1 uL which uses 13.2 ng of DNA. How many colonies would I have if I assumed a 5e6 cfu/ug transformation efficiency 5e6*"cfu"/"ug"*1*"ug"/(1000*"ng")*13.2*"ng" = 66,000 cfu 1e8 cfu -> 1.32e6 cfu

I'll just try an electroporation using 2 uL of the solution I have and see what kind of transformation efficiency I get.