Hi, all,

I am now creating a cDNA library from retina and faced some problems.

I extracted RNA from the tissue and used 2 micro gram total RNA for cDNA synthesis by using EasyChone cDNA library construction kit from Dualsystems Biotech. According to the protocol I reverse-transcribed mRNA and generated cDNA with same sequence (contain SfiI restriction site) flanking both 3' and 5' side. Late on, I performed PCR with optimized cycle number to amplify the cDNA with the primers mentioned above. Subsequently I digested the PCR product and backbone plasmid with SfiI and ligated them together (backbone 450ng, 20-80ng PCR product) 16°C for overnight. After transformation with 100 ul XL1 blue cells, there were unfortunately only around several thousands of colonies.

In principle, after transformation I must observe 10^6-10^7 colonies to reflect enough complexity of the cellular mRNA and extracted the plasmids from these colonies to create my cDNA library. However, since so far I only got several thousands of colonies, which is far away from reflecting the complexity of mRNA expression, I am quite upset about the experiment.

Is there any suggestion about the experiment?

Many thanks in advance.

Haijia