I want to make a deletion construct. My gene of interest in p3xflag cmv vector, insert size a round 1.8kb, total vector size 6.4 kb . So I want to make a construct from this vector. I want to delete around 150bp from the middle part of the gene of interest. So can anyone help me to make this construct?
Hi!
Consider the gene of your interest into three parts-first 825 base pairs, the next 150 base pairs (the region you want to delete) and the last 825base pairs.Let them be 'a', 'b' and 'c'. Design primers to amplify the 'a' and 'c' regions (take care that the forward primer of the c region has the reverse primer of the 'a' region as overhang). Now using the pcr products 'a' and 'c' as templates and forward primer of 'a' region and reverse primer of 'c' region, run another pcr. This product will have 'a' and 'c' together (which no 'b'). Now using the restriction enzymes which corresponds to the sequences used during primer designing, clone this product into your vector.
Hope this helps.
All the best!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line