I am new in cloning and likewise interested to clone 1 gene from fungal DNA (Mycelium). Now the problem is I designed primers from the complete CDS sequence of gene with 516 bp length. My primers forward is with start codon ATG but reverse with not stop codon as it is in the 3/4th part of gene. The amplicon size of gene is 389 bp but I am able to amplify the full CDS fragment ~ 516 bp on 1 % agarose gel. I have few queries regarding this as follows.
- If it good idea to proceed in this case with such primer for cloning, as the getting size is equal to I am expecting.
- Since, I designed the primers from comleate CDS and they are without RE (both forward and reverse) does it make some difference. If yes, would it be good idea to add REs now to the primers or I need to design new primers from genomic region.
- Here are my primer sequences are 5'- ATGTGGCATATCTCGAAGTAC-3' and 5'-TGGCATATCTCGAAGTACTGA -3' (without reverse completment)
Thank you in advance