about RAPD PCR of plant genomes? - (Sep/12/2006 )
Hello Members,
I am working on use of molecular markers in plant genomics. I would like to know if it is advisable to use a single RAPD primer in PCR or should we use two. The reason why i,m asking this is b/c I'm getting bands by using even a single primer. Moreover, which primers are advised to be used as reverse primers in RAPD. Are they some sort of universal primers or simply other RAPD decamers?
I am working on use of molecular markers in plant genomics. I would like to know if it is advisable to use a single RAPD primer in PCR or should we use two. The reason why i,m asking this is b/c I'm getting bands by using even a single primer. Moreover, which primers are advised to be used as reverse primers in RAPD. Are they some sort of universal primers or simply other RAPD decamers?
As I learnt (never done it on my own) you can do both: Use a single primer per reaction (length of product depends on elongation time) or two primers (product is occuring when these arbitrary primers by chance bind to DNA regions which are not too far away, so many primers are needed for trial and error). I think the first is the more common alternative.
One lecturer called this the black box voodoo techique.
P.s I would follow an established protocol, there are many available in papers/www.
Someone once defined RAPD as Random Amplifications Produced Daily, which hints at the difficulties in replication. As hobgoblin says, usually only one primer is used per reaction, so don't worry about reverse primers, but many reactions, each with a different primer, will increase your chances of finding a polymorphism linked to whatever phenotype you're interested in. If you do pursue the RAPD approach, be aware that you will always have to do things exactly the same way each time if you hope for reproducible results.
Thanks! It explains a lot. I am getting reproduceable results but they are still not what i want after using about a hundred primers.
Just pray for me!
I am working on use of molecular markers in plant genomics. I would like to know if it is advisable to use a single RAPD primer in PCR or should we use two. The reason why i,m asking this is b/c I'm getting bands by using even a single primer. Moreover, which primers are advised to be used as reverse primers in RAPD. Are they some sort of universal primers or simply other RAPD decamers?
There is no need to use any reverse primers in RAPD, universal primers are available with OPERON, UBC or Pharmacia LKB, you can also go for custom made primers. the same primer binds on opposite strands of the DNA and amplifiation occurs if the distance of the DNA betweeen the bound primers is in the amplifiable range (less than 2 Kb), so you can expect bands less than 4000 bp. score only those bands between 100 and 4000 bp. Use the same brand of polymerase and follow the same reaction conditions through out your experiment. Being dominant markers they give only half the information in comparison to co dominant markers. AFLP/SSR would be a better option. However, if you wish to improve the reliability of your RAPD markers, converting to scar markers is the best option, procedures are available in the net
wish you good luck
tes