Amplification of 3KB protein from Hela mRNA PCR PROBLEM - PCR Amplification of 3KB protein (Jan/24/2006 )
Im having difficulty amplifying a protein of 2772 bp using Hela derived cDNA. I am using Taq DNA polymerase. i have already achieved amplification of 399 amino acids from the C-Terminal at 55 degrees celcius using extention times of 1min/1KB for 35 cycles. My housekeeping protein is working too under the conditions described. the quality of the mRNA is good so i doubt thats the problem. Would appreciate advice on the cycle times/temps to use please
Is it possible to amplify from DNA or are the introns too big?
Maybe it's your taq, have you ever amplified DNA that long with your taq?
ive successfully amplified a 399 amino acid C-Terminal region but its the full length of this protein im really after.never tried a protein this long before. dont think its possible to use DNA
you may have secondary structure in the 5' end of the mRNA causing the RT to not proceed through to the full length 5' end... You may want to redo the RT or use 5' RACE kits that will address the problem of secondary structures? Cap-dependent RACE would make sure you had full length, or using high temperature RTs and higher RT temp will help denature strong stops due to secondary structure etc..
this is only IMHO...I have seen people on this forum swear that RNA should never be heated...however, my RT protocol (from Promega) using MMLV says that your template diluted in a little water and primers (I use oligo(dT)vn23) should be melted at 70C for 5 min, then do a quick-spin at 4C and put the tubes on ice...this is to help melt secondary structure...then you add a cocktail of the other components, still on ice, then put at 42 for the reaction to occur.
I have not experienced undue degradation or had any difficulties with this melting step added
To steal an "aimee-ism" Aimikins is all over this one!!! There should be no problem with a short denaturing step as long as divalent cations are not present (ie: Mg++ from the DNAse step) and this may be enough to solve your problems without the extra effort my suggestions above would have entailed... Try this first!
You could even use random nonamers to prime cDNA synthesis after heating as this will also improve the probability of RT-ing to the 5' end...
In the experience of our lab, people used to indeed heat the RNA for 10 minutes @ 65°C, then cool it down rapidly and keep it on ice untill the RT reaction mix was added and put on 42°C for 1 hour.
right now I do my RT with an enzyme @ 55°C, so I no longer need to heat it before the reaction because the secondary structure is denatured enough.