MOLECULAR TEST WITH !JUST PCR AND GEL! FOR A STARTER - PLEASE GIVE ME SUGGESTIONS (Aug/15/2006 )
IN OUR LAB WE HAVE
2 PCR (GRADIENT PCR BUT NO REAL TIME PCR)
1 COMPUTERIZED GEL IMAGING-ANALYSIS SYSTEM (AND SOME OTHER STUFF LIKE MICRO SANTRIFUGE...)
I DECIDED TO TRY SOME MOLECULAR TESTS TO GET USED TO MOLECULAR DEVICES AND LABORATORY.
I'VE BEEN DOING MANUAL DNA ISOLATION (PHENOL-CHLOROFORM) FOR A TIME WITH SUCCESS.
WHAT DO YOU SUGGEST ME FOR A STAR TEST WITH JUST PCR - GEL AND IMAGING-ANALYSIS SYSTEM. (WE CAN GET PRIMERS AND RESTRICTOIN ENZYMES ANYTIME WE WANT).
have you looked at your DNA on an agarose gel yet or quantified it?
what are you purifying DNA from ??
is always useful to know background of the techniques used in case of problems...
why not choose primers that have been used successfully in the past and have a go a optimising the reaction conditions from scratch......, can have a go a different pcr`s (normal, hot start, touchdown etc.)
once you have mastered that you can then continue to subclone your dna and play around with some restriction enzymes, then clone,express and purify the protein if you want to go that far ahead....
hope this helps!!
I have quantified with agarose gel and also looked for OD with spectrophotometer (mean OD was 1.65-some phenol contamination I think)
we have been working on cytogenetics nearly for a year and we havent done any pcr reactions. (we've just activated our molecular genetics part) Because of this I wanted your suggestion for a good start to molecular genetics.
what is the best test for a start (diagnostic or not) can be done just with pcr agarose gel and gel imaging (restriction digestion I think).
thats sounds fine, though digesting genomic dna will only show a big smear on a gel (if your lucky)
not a very nice picture for your lab book!!!!
why not try and amplify a gene by pcr, have a look on pubmed or somewhere like that (one that has been sequenced), and restrict that as you can calculate the relative size of the fragments that you should be able to see.
you can still play around optimising pcr conditions which will aid understanding somewhat