I want to amplify the cDNA sequence of a large gene in zebrafish (around 10kb). I am looking for any tips around RNA extraction protocol and cDNA synthesis so that my whole cDNA fragment isn't fragmented. Does anyone have any suggestions?
Many thanks in advance!
Jeroen
Trizol for RNA extraction first, followed by Superscript RT using random primers. This will work fine.
Definitely the random primers would be the way to go for the RT. It is also very important to use a pcr system designed for long range (with enhanced fidelity and processivity) such as Phusion from Thermo, LongAmp from NEB, KAPA Long Range, etc. This will ensure that your product would be less likely to contain pcr errors.
You have not told us what the intended use of the cDNA would be. If you need it to produce a product (protein expression, etc) then you need to ensure that there are no mutations introduced by the RT and pcr steps.
I would strongly suggest considering the longer route of amplifying "chunks" of the cDNA and splicing them together using a restriction enzyme approach. For eg: analyse the sequence and look for unique cutters that will split it into 2-3 roughly equal sized sections (2-5kb each). Design primers to amplify those and clone them. You can then make sure that each section is error free by sequencing the clones and then you can stitch them together using the unique RE sites. You are going to have to design internal primers for sequencing regardless so you might as well start with this approach in mind.
Thank you for your answer David.
Our application is indeed protein expression. We have Trizol, Superscript RT, Random Primers and Phusion from NEB.
In the past we were unable to ligate separate 5kb cDNA fragments to each other, so this is why we want to try to PCR it as a whole. Finding the right RE sites is also hard due to the fragments being so long.
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