Amplification of 3.4 kb product from RNA for cloning - (Dec/26/2010 )
I am trying to amplify the complete coding region of a particular gene of 3.4 kb size. I want to PCR clone it into an IRES2 -gfp cloning vector. To the primers again 15bp homologous sequences have been added. The Tm of bothe the primers minus the 15bp homologous bases is around 56 deg. GC content is 65%. I started with 5ug/ul RNA and used Titan One step PCR Kit from ROCHE which contains AMV RT as well as Hi-Fi Polymerase and can amplify upto 6kb long product. But still Im not getting any bands. Can any one help me?
It's the Tm of the region matching the template (homologous region) that matters. This likely needs to be longer than 15 bp for normal PCR conditions. Did you really mean that 15 bp were added to the 5' end of a primer otherwise matching a template?
I used Clontech infusion primer design software where infusion primer design tool converts the existing primers (which in my case was 20 mers long with above mentioned GC content and Tm)into infusion primers by adding vector specific sequences (15mers long)on the 5'side of the primers.So then the modified primer becomes 35bp long.
So what Tm shd I consider? Inclusive of this extra bases (which the clontech protocol says u dont have to). I really dont know. I am not able to amplify my gene of interest using these primers.
Your initial pcr cycles will only bind the primer with the 20 bp of template matching primer DNA. That's the region that will control your initial Tm. But usually people worry FAR too much about Tm. 55C works most of the time for pcr annealing. If it doesn't, then there is often something else wrong.
Should I try taking large amount of template (5-10ug/ul) as a starting template, will that help?
Did you analyze the expression of your gene of interest in the sample you are using (for example by realtime PCR)? Is your gene of interest expressed in sufficient amounts? Sometimes reamplification of your pcr product using the same primers helps to get a product if expression is low. But you may encounter mutations in your insert due to misincorporations (even a proofreading polymerase is not 100% perfect)
Doing two step RT-PCR I also often had problems to amplify larger fragments (>1 kB) from cDNA. In my opinion the cDNA one gets after reverse transcription is kind of fragmented, making amplification of longer products difficult.