Hi,
I need to make a construct for protein localization. The construct should be made of the gene of interest and the GFP gene. I don't have idea on how to stick the fragments together with the PCR approach . I understand that the first PCR cycle should generate fragments with overlapping ends and the second PCR cycle should fuse these fragments together. I can't figure out how this works. Can you give me some drawings or images that helps me understand? Also, I need to insert restriction sites because I need to insert all the construct into a plasmid..In which stage and where in the construct do I put the restriction sites?Can you explain to me?
Many thanks
HI,
There are several commercial GFP, YFP or other fusion tag plasmids in the market. It is easy and efficient to tag protein of interest.
Design your POI primers with suitable restriction sites and digest the plasmid in region so that the tag remains in the frame. You can check if expression works in animal cell line and you are good to go.
Santosh
1. Santosh's suggestion is one way to go. If you have a plasmid with GFP already, you can just stick your gene-of-interest (GOI) next to it (in-frame, no stop codon in-between).
2. As you asked how to fuse 2 fragments together (GOI::GFP) with PCR approach (then stick into a plasmid):
See attached paper (assisted with In-FutionTM kit), it has a good figure to describe it too.
My previous answer (point '2'), it uses a In-FutionTM kit.
If you are fusing 2 fragments together (GOP::GFP) solely using PCR method, without using any commercial kits (looks like you are doing so). Please see this discussion on RG: [especially, April Pawluk's answer]
How to join two or more fragments using primers?
https://www.researchgate.net/post/How_to_join_two_or_more_fragments_using_primers
Hi Silvia,
We have found in our transgenics production service that donor homology plasmids can easily be assembled using Gibson ligation technique. Basically all components of a plasmid can be turned into PCR products. Make sure each PCR overlaps at about 30 bp with the adjacent PCR. We routinely use this for up to 5 PCR parts in a plasmid assembly reaction. We are now into the 1000s for plasmids assembled by Gibson ligation technique. Average number of PCR parts is 3.
One good source of Gibson reagent and more information on its use NEB:
Chris
https://www.neb.com/products/e5510-gibson-assembly-cloning-kit
- Badges
- Science method