Hello there, I would like to ask the community regarding on the work I'm conducting;
1) How do I use the Primer3 (http://frodo.wi.mit.edu/primer3/) online or other freeware to detect the repeat motifs from obtained sequence? I have tried on the first column where I have inserted the sequence based on the chromatogram pattern, then I checked the pick left and pick right primer box, then I just simply press pick primers. Or do I need to set few parameters upon primer picking? If yes then what is the basis for it?
2) After detection, the process should be synthesizing the primer pairs then validation by re-amplify it with the DNA used in the 5'-anchored PCR. Some papers are going beyond this point by labelling it either with radioactive or fluorescent dye. What is the purpose of labeling the 5'-end primer that will flank microsatellite repeat motifs?
3) My lab has run out of competent cells but we still have the TOPO TA (Invitrogen) vector. How do I produce chemically competent cells that match with the vector? Do I need to identify the E. coli strain that fits with the vector?
4) What is the purpose of doing colony PCR? How do I do it?
Thank you very much