I want to make cDNA library from RNA. I am using QIAGEN kit to get a cDNA, however the product obtained is single stranded cDNA. Can anyone suggest me how to convert this single strand cDNA into double strand cDNA. or Any other method to obtain cDNA library.
Please suggest, any help is highly appreciated.
Thanks
Thermo Fisher and other companies offer kits to get double stranded cDNA:
https://www.thermofisher.com/order/catalog/product/11917010
@Dennis thankyou for the reply, can you suggest alternative method, because, this kit is very costly, I have atleast 100 samples for analysis and this means 100 reactions.
I have not much experience with cDNA. I just use it for qRT-PCR to check my gene knockdowns.
But isn't it just possible to do a normal PCR with the cDNA to get double stranded cDNA? Or at least you could do all the steps from a classical PCR once without the denaturation part at the beginning (because it is already single stranded). Maybe this could work. But I have never done it and it is just an idea. But maybe it is worth a try if nobody comes with a better idea :D
Wish you all the best and good luck.
2nd Strand synthesis with dUTP from First Strand cDNA:
10xNEB buffer 2 1.5 uL
10mM dUTP mix 1 uL
RNaseH 0.2uL
DNA Polymerase I 1 uL
Purified First Strand cDNA 10 uL
H2O 1.3 uL
16°C, 2.5hrs
Thank you Jose for your answer. But, don't you need to ligate 2nd strand fragments? Would you share detailed protocol.
Hi all, I want to do whole genome sequencing of DENV with Oxford nanopore would like to know if do the steps am using make any difference or sense
1. Extract total RNA from blood
2. Confirm presence of dengue RNA with universal RT- PCR primers
3. Identify serotypes with serotyp-specific RT-PCR primers
4. Back to step 1 then 5
5. Synthesis of double stranded cDNA using a set of serotype specific oligo (dT) primers that would scan through DENV genome to make about 5-7 fragments of ds cDNA
6. Purification of ds cDNA
7. library prepration with rapid sequencing kit (ONT, manufacturer)
8. Sequence run
Will it make sense to substitute serotype-specific oligo (dT) primers with random primers (hexamers) in step 5
Any comment?
Hi Abhishek Tyagi , Nikola Kotur
Did either one of you find the solution? Thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA