Problem amplifying 3 kp fragment from a plasmid - (Jul/29/2005 )
hi.
I have tried amplify the DNA genome of plant virus. the genome is cloned in plasmid vector, but i designed primers with restriction sites because i need amplify the complete genome in order to clone it in other vector with specific restriction sites. Then I´ve been doing PCR with the specific primers, the fragment amplified must be 3000 pb. the firs time i had luck. but after i tried newly but i don´t have had luck. the master mix used was used in order to amplified other fragment and the fragment was amplified. So I don´t know that to do.
3000bp is quite a large fragment for conventional Taq to amplify, are you using normal Taq? or a formulation for long PCR, such as Expand Long Template from roche or equivalent?
If you arae using conventional Taq, what is your extension time? usually allow 1min for each KB of template.
Good luck!
Nick
What enzyme are you using? As said, not all enzymes are capable of amplifying 3000 bp.
Apart from that, have you changed annealing temperature, Mg concentration, primer concentration? You will be surprised what difference these parameters can make (I am doing pcr's that work with an annealing temperature of 55°C and no longer give any product with an annealing of 57°C for instance).
I have tried amplify the DNA genome of plant virus. the genome is cloned in plasmid vector, but i designed primers with restriction sites because i need amplify the complete genome in order to clonar it in other vector with specific restriction sites. Then I´ve been doing PCR with the specific primers, the fragment amplified must be 3000 pb. the firs time i had luck. but after i tried newly but i don´t have had luck. the master mix used was used in order to amplified other fragment and the fragment was amplified. So I don´t know that to do.
It's a bit difficult to understand what excactly is the problem.
Let me guess: Your plasmid vector (with cloned 3000 bp genome) is transformed into an E.coli. You extract the plasmid, and try amplifying the genome with plasmid as template? Right?
In that case, you could try doing a colony PCR from a very fresh plate. That means streak out your Transformed bacteria on a selective plate. The next day, make a PCR mix with your specific primers but without template. Take a yellow tip and dip it into one colony (just dip once, not take a lot), rinse the bacteria of the tip into the PCR mix, and imediately start the PCR.
Why not extract Plasmid? - sometimes just waste of time, and sometimes PCR works better from a colony. The important part is having a fresh colony.
Now, I might have understood your problem wrong. Other fixes might be freshly extracted DNA, and not too much of it. Sometimes PCR just do not work with stored DNA, and it's quicker to do a fresh isolation of DNA than to clean up the DNA (if, as in your case, you are working with a cloned template).
The other possible problem solvers, if you are sure the template is not the problem, are: heat the nucleotides 20 min at 65 C before use, fresh tube of nucleotides, fresh tube of everything else, good luck .....
Hi anybody
I want to thank you for advices.
I`ll take in mind all of them.
Thank´s again
dtrejo.