How isolate a 4kbp gene?! - (Jun/21/2007 )
Hi everyone,
I want to isolate a 3.6 kbp gene from Arabidopsis via CDNA library and swing PCR technique.I have some question about It.Please help me through this 
- Can I do that with PFU ? I heard that it is very hard to work with this enzyme. if anyone have suggestions?
- Is it possible to extract an intact (non degrade) 3600 bp mRNA and reverse transcribe with RT?
- and about sources ( books , articles or websites) . Do you have one of these about my project? please introduce to me .
I will be very glad if someone that done a parallel project , share his/her experiences with me.
I appreciate any suggestion or advices.
Thanks 
Hi,
Personally, Phusion(Finnzymes) or Iproof(BioRad) is better than Pfu, in synthesis speed and fidelity. They are also easy to handle. the former does the tricks of high GC content.
Someone has isolated 23kb mRNA, 3.6kb RNA should be easily isolated if highly affluent, Trizol Method plus GTC is very efficient. Life Tech Focus has published this method. you can refer it. Of course, you can also buy a cDNA Pool including your gene, If thus, you can save a lot of time.
Best Regard, and Good Luck.
Cinba
I want to isolate a 3.6 kbp gene from Arabidopsis via CDNA library and swing PCR technique.I have some question about It.Please help me through this
- Can I do that with PFU ? I heard that it is very hard to work with this enzyme. if anyone have suggestions?
- Is it possible to extract an intact (non degrade) 3600 bp mRNA and reverse transcribe with RT?
- and about sources ( books , articles or websites) . Do you have one of these about my project? please introduce to me .
I will be very glad if someone that done a parallel project , share his/her experiences with me.
I appreciate any suggestion or advices.
Thanks
hai
i have worked with pfu. but the problem is u have to standardise the amt. of enzyme to be used. i tried standardizing it with a combination of taq. a concentration of 1: 100 worked for me.
Hi, thanks a lot for your advises.