primer design

Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies

Moderators: r.rosati, mchlbrmn

primer design

Postby VISHAKHA SHARMA » Feb 06 2017 4:24 am

I a master student and I have to design a primer so that of the stop codon sequence is replaced by the coding sequence and the two sequences get ligated together
VISHAKHA SHARMA
newcomer
newcomer
 
Posts: 1
Joined: Feb 06 2017 4:11 am

Re: primer design

Postby relaxin » Feb 06 2017 12:27 pm

I do not understand what you want to do. Please give us more details.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7174
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: primer design

Postby 29yrsExperience » Aug 29 2017 8:09 am

Here is a general answer to your undetailed question: Sounds like you want to make a gene fusion. It will really help you to use some kind of plasmid design software. In your software start with the coding sequence(s), stick them together, change the stop codon on the first one to some other codon (usually some neutral aa residue), and add an appropriate restriction site for joining the sequences together. You can pick a codon that forms part of the restriction site. If it is a R-enzyme site that can be overlapped by a dam or dcm methylation site, (and the enzyme is sensitive to methylation) avoid creating those methylation sites. Then, check whether it will be in-frame with the second coding sequence by translating it. You may need to add nucleotides (1 or 2) to get both sequences to translate they way they should. Once you get something that works "in silico" design your primer based on that new junction sequence.
29yrsExperience
technophile
technophile
 
Posts: 34
Joined: Aug 11 2017 12:58 pm


Return to DNA and General PCR Methods

Who is online

Users browsing this forum: No registered users and 1 guest