cDNA Library Construction by using kits and Sequencing - (Aug/04/2014 )
I have started to study master of science. In my lab, venom peptides are study area, and I will carry out its transcriptome part as a molecular biology graduate. I have some experiences about RNA isolation, cDNA synthesis and qPCR. However, I need to perform cDNA library construction from venom gland and sequence it. Unfortunately, Neither me or someone else from our lab have no experience about it. I will write general steps that I need to apply, and questions & concerns for each step.
1. Total RNA Isolation
-> Since I am interested in expression of venom peptides of my sample, I need its mRNA sequences. I wonder that is it necessary to purificate mRNA from total RNA, or is it still okay to use total RNA as a template for cDNA synthesis by using oligodT primers (I think it complements only to poly A tails, so amplify only mRNAs, am I right?)
-> Which kit should I use?
2. cDNA library construction
-> first strand cDNA synthesis
-> PCR for enriched full-length ds cDNA
-> Ligation into vector ( which vector should I use?)
-> What about SMART cDNA library construction kit? (price, efficiency, application difficulty etc.)
4. Plasmid Isolation
What about alkaline lysis method?
5. Sequencing and obtaining high quality ESTs
What about sequencing process? 100-150 ESTs, hope that some of which may be novel peptide candidates, are enough for me. So, to obtain it which sequencing method is best for me, and which requirements have to be supplied by me to personnel who is responsible for sequencing?
For a researcher who has no experience about these techniques, do you think how long does it take to apply whole procedure? Is it logical to attempt this without experience? What should I read, watch, follow about this issue??
I will be very pleased to any comment or any guidance.
1: It should be OK to use total RNA. Note that not all mRNA are polyA tailed, but RNAs other than mRNA aren't usually polyA tailed, so this will enrich for the majority of mRNAs. Kit: depends on the sample, the gold standard is still Trizol extraction.
2:Vector - use something suitable for your purposes... what do you plan to do with the cloned sequences? I don't know anything about the SMART cDNA kit.
4: All the common kits etc use alkaline lysis for plasmid extraction. It works well, and is easy to do, even if you want to make up the buffers yourself and do a traditional plasmid DNA extraction.
5:You might want to consider a next generation sequencing process to tell you levels of expression of the particular mRNAs as well as sequence information. I don't know if these platforms are available for your tissue types though. The reagents etc that need to be supplied by you depend on who you are getting to do the sequencing.