I would like to share my today's experiment gel image for cDNA purification.

The Lane 3 corresponds to purified cDNA amount (purified through chromo spin column TE-400) while lane 5 corresponds to unpurified amplified cDNA sample.

Details I am giving you as following:-

Total 2 micro gm total RNA of plant root tissue was used to prepare cDNA as described in Y1H manual, followed by Advantage 2 PCR kit used to amplify the cDNA that would be considered as unpurified , today I have checked the concentration for both unpurified and purified sample (passed through column) through nanodrop.

The nanodrop result was as following 

Purified cDNA - 55ng/ul and total volume of my cDNA sample is 20 ul.  (total purified amount= 55x 20= 1100 ng)  {after purification of unpurified sample through kit column, each column was used for 100 ul sample purification as limited capacity, finally after passed through column total 200 ul sample at the end was used for c DNA precipitation step through potassium acetate} 

While concentration of Unpurified cDNA- 500ng/ul and total volume of my sample was 100 ul.

On gel the loaded amount was around 75 ng sample (1.5 ul volume) was checked for purified sample while for unpurified around 2500ng sample (5 ul) was used to check.

Can you please respond after looking at my attached gel image.

May I proceed with my library preparation analysis for Yeast one hybrid with this sample that I have around now 1000 ng  cDNA in amount instead of using 2-5 micro gm as suggested in manual.

Is it possible that nanodrop does not give correct estimation for cDNA sample as I assumed through image?