1. What is the reason I prepare High, Medium and Low molecular weight bands from the cDNA-Gel? 2. Why is it important to add the bar code primer to the targets before library amplification instead of during library amplification?
Hello Christoph,
(i) Sometime I have noticed that the dominant species of band in the low molecular weight band from the cDNA gel is primer dimers, this can ruin the complexity of you cDNA library and if that is the case it would not be advisable to sequence the lower molecular weight band. Also, it checks the fidelity of your final PCR. Amplified products should fall within the same molecuar size region as the gel slices you cut.
(ii) By adding the bar code primer to targets before library amplification I guess you are reducing the likely hood of amplifying any cross contaminating RNAs later on in the preparation of the libraries.
If you need more help with this you should view these two pages:
https://docs.google.com/document/d/1qSMMjwvFtytVcmyAgcLrktgE2KjMuLK55f1oCcexaWw/edit?hl=en_US
http://ulelab.info/publications/Konig_wiley.pdf
Hope this helps!
Hi Ross.
Many thanks for your helpful reply. How do you confirm that the lower band consists of primer dimers? By co-running the PCR blank?
Cheers.
yes you can do that. Or the primer dimers will run just below 150bp.
Hello Christoph,
1) For the first question, I don't know.
2) You need to use the bar code to determine wether a read is a PCR duplicate or a unique cDNA product. So it need to be added before PCR amplification.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA