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
Hi,
Just a quick answer to your last 2 questions.. When making competent cells you should make sure you pick a methylation-recombination-restriction deficient strain, I guess you could use DH5a or DH10B strains which give high yields and are also suitable with the vector. The TA vector has a ColE1 rep origin which I think can be used with any E. coli strain, perhaps with more or less efficiency. For making chemically competent cells you could use the CaCl2 wash method, or the methods devised by Hanahan or Inoue -look at the Molecular Cloning Manual, or in the net:)
When you do colony PCR you are basically trying to see if your cloning/transformation has worked, avoiding doing plasmid prep first. Basically you pick a colony, throw it in PCR water and take a small amout as template for PCR with vector or insert specific primers. What you should watch out for is the false positives, there can easily be a bit of the ligation mix or vector sticking to your cells, to the agar or anywhere, so if you have + PCR results may not always mean that you actually have the vector inside the cell. There are ways of avoiding this by doing some washing steps, but you might as well do plasmid prep:))
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