Newbie- choose DNA template - (Jul/01/2011 )
Hi. I am newbie at PCR and I have a question. I am planing to construct two GFP-tagged human proteins-producing plasmid. So, my plan is 1. prepare PCR products containing DNA for target proteins 2. put PCR product to GFP-vector. 3. transfect the PCR product-inserted vectors to appropriate cell lines. To do so, I found the DNA sequence for my target proteins and designed primers for PCR. But, I don't know exactly how to prepare DMA templates for the PCR reaction. How can I have DNA template for my target proteins to perform PCR?? DO I have to purchase or prepare human genomic DNA??
Thanks in advance for the tips
First you have to answer a question
Do you plan to work with cDNA (without introns) or a full size gene?
Human gene tend to have splice site variant and contain regulatory elements within their introns. A cDNA will not be able to capture these facets.
On the other hand full size eukaryote tend to be very big and not easy to manipulate. cDNA tend to be much smaller.
Depending on your answer, you may use one these as a template
a plasmid from a cDNA libraries
a plasmid from a BAC libraries
raw human DNA.
So where can you get a template?
You may either purchase the template DNA or obtain it from a lab working in the field. Using pubmed find out who has publish work on your protein of interest and email them. Invitrogen and Open Biosystems are examples of places where you may purchase a BAC libraries. A google search will turn up more places which sells cDNA or BAC libraries.
Working with raw human DNA is not easy. Stick with libraries.