Notes+on+calculations+of+concentrations+from+gel+images+4-11-13

-Total Intensity of band 3rd from bottom (300 bp) 40591 10 uL ladder was loaded so this band corresponds to 16.5 ng

Conversion factor: 16.5*ng/40591

cDNA 608801

608801*16.5*"ng"/40591= 248 ng This was in 1 uL so 248 ng/uL 4-11-13 I currently have 9 uL left Total amount electroeluted is about 248*"ng"/"uL"*10*"uL"=2480*"ng"

--Approximately how much was there originally (3-26-13)? Lanes 1,2, and 7 were used at this step. Opened Gel 1 Total 35 cycles "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\3-27-13 cdna library\gel 1 total 35 cycles 1hr 70V 2p0 exp.jpg" Cumulative Intensity of 3rd from the top band (12*"uL"*500*"ng"/(20*"uL")*36*"ng"/(500.0*"ng"))/26894 = 21.6*"ng"/26894

Cumulative intensity lane 1: 859941 859941*21.6*"ng"/26894= 691*"ng" total

Cumulative intensity lane 2: 794374 794374*21.6*"ng"/26894 = 638*"ng" total

Opened Gel 2 total 40 cycles "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor cDNA Library\3-27-13 cdna library\gel 2 total 40 cycles 1hr 70V 2p0 exp.jpg"

Cumulative intensity of 1 kb band: 30047 Conversion factor (12*"uL"*500*"ng"/(20*"uL")*108*"ng"/(500.0*"ng"))/30047 = 64.8*"ng"/30047

Cumulative intensity lane 7: 517848 517848*64.8*"ng"/30047 = 1117*"ng" total

lanes 1, 2, and 7 were combined so there should have been a total of 691*"ng"+638*"ng"+1117*"ng"= 2446*"ng"

Estimated Percent Recovery: 2480/2446*100.0=101% So the estimated percent recovery is close to 100%

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-Purified Ladder 1 kb band 29683

29683*16.5*"ng"/40591=12.1 ng The 1 kb band is 108/500=0.216 of all the material so there is a total of 12.1*"ng"/0.216=56.0 ng

so 56.0 ng/uL There was 10 uL so 56.0*"ng"/"uL"*10*"uL"= 560 ng

I originally (ON 3-22-13) added 30 uL ladder which corresponds to 30*"uL"*500*"ng"/(20*"uL")=750 ng

Estimated Percent Recovery 560/750*100.0 = 74.7%

- -Linearized Plasmid (50*"ng"/"uL") 26189

26189*16.5*"ng"/40591= 10.6*"ng" so 10.6 ng/uL There was 10 uL so 10.6*"ng"/"uL"*10*"uL=106 ng

I originally (3-22-13) loaded 10*"uL"*50*"ng"/"uL"=500*"ng"

Estimated Percent Recovery = 106/500*100= 21.2%

-Control Insert (40*"ng"/"uL") 30263

30263*16.5*"ng"/40591=12.3*"ng" 12.3 ng/uL 12.3*"ng"/"uL"*10*"uL"=123*"ng"

I originally loaded (3-22-13) 10*"uL"*40*"ng"/"uL" = 400*"ng"

Estimated Percent Recovery = 123/400*100=30.8%