1. What is the reason I prepare High, Medium and Low molecular weight bands from the cDNA-Gel? 2. Why is it important to add the bar code primer to the targets before library amplification instead of during library amplification?
(i) Sometime I have noticed that the dominant species of band in the low molecular weight band from the cDNA gel is primer dimers, this can ruin the complexity of you cDNA library and if that is the case it would not be advisable to sequence the lower molecular weight band. Also, it checks the fidelity of your final PCR. Amplified products should fall within the same molecuar size region as the gel slices you cut.
(ii) By adding the bar code primer to targets before library amplification I guess you are reducing the likely hood of amplifying any cross contaminating RNAs later on in the preparation of the libraries.
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