PCR amplification problem

[bam pandit]

I have to amplify a gene of size >=3.5 kb, from an unknown genome.

my limitations:

using the usual taq
i'm using the cDNA thats 6 months old (from RACE kit)


how do i get the amplification??!!?

[danfive]

I would go straight to a TOPO vector.
I did a couple projects using them and have no complaints.
Also the kit was only a couple hundred bucks, not expensive at all (big time-saver).

[bam pandit]

dear danfive, thankyou for your reply, but the problem is that i haven't yet reached the stage of cloning, i'm yet to know why i cannot get the pcr product right... all i get to see is the dye or smear in the gel.

so any suggestions??

[Priyanka]

I think its written in literature that normal taq amplifies fragments upto 2kb properly. However I had a gene that was also 3.5kb long which i could amplify easily with usual taq. You could try ussing Pfu polymerase (amplifies bigger fragments, but leaves blut ends..), also the other problem could be the primers.. please check the primers properly..(bioinformatically.....there are many hitches there..)..also other Thing could be degarded DNA but I think you would have ruled that possibility out already...
Let me know when you get the amplifiaction ... and the reasons behind it.. I am really curious about this one...(Could help me also..)
Cheers
Priyanka

[bam pandit]

thanks priyanka... i'm chking the cpndintion of the cDNA... its an interesing problem for me too... i serched and there are a few othe taqs that can be used for longer fragments ... genaxxon taq, kapa2 taq.. etc.. i'll let you know he details afetr the amplification.. ... and blunt ends is what i want

[Priyanka]

I hope that you dont forget to get back with the details...
I thought may be I could Ask you for a favor... iam isolating RNA these days.. At first the RNA was completely degraded... But today the gel shows 4 RNA bands and smearing between the bands..( i think its not the best quality RNA) I would like to know some precautions/ tips that I could follow for the same.. I tried IRIS the first time.. today however i used Trizol...

[bam pandit]

hey, sorry for the late reply... you are on the right track i should say. but whats the source of your RNA? is it plant RNA or animal...?? if you can send over a gel picture, i'll be able to analyze it in a better way. good luck.

[oBWhat]

Hello Priyanka,
the smearing either sounds like bad RNA preparation or degradation in some steps.

Have you tried any RNA isolation kits ie QIagen or wizard?

[Priyanka]

I am working with plants... and Intrestingly trizol is giving little better results as compared to IRIS..( I have no idea why).. and kits are not something that my teacher recommends.. so they are out of question... the problem is still that Iam getting diffused bands of almost all sizes but along with a lot of smear.... And what about your gene (3.5kb)

[Priyanka]

I just thought i`ll let you guys know I got beautiful RNA profile with no background or anything, Perfect amount of smear (mRNA) and needless to say I am really relaxed now..
The problem I could identify was very bad Agarose quality...and bad ethanol...Thankx guys for all the help..All the Best!!
Cheers
Priyanka