Is their any maximum limit for an insert size in terms of nucleotides for mutagenesis? My insert size is 14nt and plasmid size 4.2kb so will their be a problem?
Hi Shubhashish,
14 bases should not be a problem. We have generated constructs with larger inserts several times.
I hope this helps. Good luck with your cloning!
I dont know which method are you using for mutagenesis, but changing or inserting 14 nucleotides is not a problem. I have done this both by Kunkel mutagenesis and by quickchange mutagenesis. It should also be possible with overlapping PCR.
Smaller inserts tend to be more efficient. 14 nt should be fairly efficient, depending on the mutagenesis method you're using!
Thank you all
Can you please refer me any related protocol for PCR mediated site directed mutagenesis for this insertion.
This is what I have used: https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publications