Hi im on my final project for my thesis and i get stucked in cloning phase. My goals is to inserting one gene to vector who has another gene too. So i have a gene called llm2-mutan and llm1 who's completely ligated with pet28a(+) as my vector/backbone. I'm trying everything like ligating in 16 C overnight, 22 C 3h, etc. My gene llm2-Mutan was a pcr product. Everytime i checking my colony using PCR with electrophoresis is always showing 1100 bp, but my goals is 2100 bp because i must insert 2 gene in one vector. I hope someone can helping me with my issues. Thank u
You haven't provided enough information to troubleshoot. What enzymes are being used in the cloning (both for the original gene and the new two gene construct). Are the PCR primers from flanking regions away from the cloning? Did you do controls to be sure that you had good digestion of the recipient plasmid? Are you sure you have enough insert DNA and that it was thoroughly digested?
My experience is that the problem is usually something with the DNA and rarely with the ligation conditions.
This is my double digest Plasmid and my insert in electrophoresis
Line 3 - 4 : Double digest Vector using EcoRI and XhOI
Line 6 : Double Digest my Wild type insert gene (LLM2-WT) using EcoRI and XhOI
Line 7 : Double digest my insert mutan gene (LLM2-Mut) using EcoRI and XhOI
Line 8 : Single digest LLM2 Mutan using EcoRI
What i am doing with my mutan insert gene is to change the EcoRI restriction site using overlapping PCR methode. Im using T7 promotor and Terminator as a PCR primer with anneling temperature 55 C in 30s and extension 72 C in 2 minutes using Powerpol Mix. after that im using T4 DNA Ligase thermofisher to ligating my gene.
I am perplexed by your gel, but maybe I am not understanding it correctly. You write that lanes 3 and 4 are vector digested with EcoRI + XhoI, but the band shown is much much larger than what I would expect from adding the bands in lanes 6-8. Secondly why is the "vector" band in 7 so much smaller than that in 6?
Lanes 3 and 4 are my vector (plasmid) which have a size 6469 bp before double digest . The size of my vector after double digest with EcoRI + XhoI is 5754 bp. Otherwise lanes 6-8 more smaller than lanes 3-4 because they are my insert (PCR product), they have a size around 1000-1100 bp. I'm doing a mutation for my insert using overlapping method with PCR to change the EcoRI restriction site. The wild type insert which is lanes 7 have a restriction site EcoRI in the middle of the gene. So if i digesting Wild type gene using EcoRI, my sequence gene suddenly get digested too. otherwise lanes 8 is single digest LLM2-Mutan whos undigested by EcoRI and have a size 1100 bp. Its different with Lanes 7 LLM2 Wild type whos get digested using EcoRI with a size 891bp.@ The lane 6 is double digest LLM2-Mutan using EcoRI and XhoI (1000 bp)
- Badges
- Science topic