How to remove copy of redundant cDNA in a mixture - RT results in a cDNA library with high copy number of few genes (Feb/18/2010 )
I want to sequence a cDNA library made from mRNA extracted from a specific tissue. I do not plan to sequence whole library but just want to find some unknown genes expressed in that tissue. Therefore, I added to each cDNA a primer (through reverse transcription) and 1 adapter (using T4 RNA ligase). Then I amplified all using 2 primers corresponding to the primer and adapter mentioned above. The amplified product (very high yield, after purifying, 350 ng/µl, ranging from 100 bp to 5 kb in size) was cloned into TOPO TA pCR4 vector and 200 clones were sequenced with T3 and T7 primers. I found that only few genes were inserted during the transformation. They would had high copy numbers in the mixture of cDNA.
Do you know any way to remove redundant RNA or cDNA in the library so that other less abundant ones can be equally cloned?
Other ideas also be very appreciated.
I understand that your purpose is to remove redundant clones and sequence only the rare transcripts. I suggest you to go through clontech differential screening kit manual.
All the best
As i felt that both of us are working on related topics. i felt it is better to clear my doubts with you.actually i have constructed CDNA library from a plant species. every thing appeared right till TA cloning and colony PCR with M13 primers. but no plasmid observed after plasmid isolation . dont understand whats the problem with this since one and half month . hope some one helps me.
THanx a million tons
Thanks for your kind replies. I have sequenced my cDNA clones, about 300 clones first and found that not many genes with abundant copy numbers present in my library. Therefore, what I will do is just isolate only appropriate size range of cDNA and clone fragments in that range.
In your case, it may be necessary to know the protocol you used to culture (medium, antibiotic concentration, temperature, shaking, and incubation time) clones for plasmid extraction. Once I also failed to get enough plasmid. To my experience, increasing ampicilin concentration to about 300-350 µg/ml helps to improve plasmid yield and you can have enough plasmid form 1.0-1.5 ml of culture. In addition, shaking at 500 rpm at 37ºC for 16-18 h are good conditions.