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I am posting questions that student’s have emailed me and my responses in case you may have the same question: Q:Primer Waliking-- that is when you use two primers going in opposite direction, and it is used when an insert is to big to sequence normally with the use of one primer. Is this correct? what is considered big I have 600bp, and 5000bp. A:YES AND NO. PRIMER WALKING IS DONE WHEN THE INSERT SIZE IS LARGE (GREATER THAN 5000 OR SO). YOU DESIGN A PRIMER TO A KNOWN SEQUENCE, DO A SEQUENCING REACTION TO DETERMINE SOME UNKNOWN SEQUENCE, THEN DESIGN A NEW PRIMER BASED ON THIS NEW SEQUENCE INFO. THEN YOU DO ANOTHER SEQUENCING REACTION, ETC, ETC AND "WALK" ACROSS THE INSERT. YOU CAN "WALK" FROM BOTH DIRECTIONS, USING TWO, SO THAT YOU MAY MORE QUICKLY DETERMINE THE ENTIRE SEQUENCE. YOU CAN ALWAYS USE TWO PRIMERS AND GO FROM OPPOSITE DIRECTIONS, EVEN IF YOU ARE NOT PRIMER WALKING.
Q:This next question is on the lab part. The DNA fingerprinting experiment-How was the technique different than what would be done normally? Are you looking for how we did our exp. as to a southern blot. We used paper towels, thw weight was a beaker, saran wrap.. Is this what you mean? A: TWO MAIN DIFFERENCES, BOTH RELATED TO PROBING 1. WE USED COLORIMETRIC PROBING RATHER THAN RADIOACTIVE PROBING 2. Bigger difference: WE DID NOT USE A NORMAL SSDNA PROBE, BUT HAD BIOTINYLATED DNA ON BLOT PROBED WITH SAAP |