Hello,

could you please specify the question further to get a good answer? For example, is there some particular problem with the amplification or are you considering the best kit to carry out the amplification?

Hi Eero,

Yes, I have cDNA amplified with that kit but cannot get the PCR from my cDNA to work. So I will start over from RNA I know is good quality. So, Im woundering if there is any reason I should not use Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit? The protocol says one can obtain up to 11kb but I wonder if anyoone has any experience with the kit and large fragments. The gene I later want to amplify from my cDNA to clone in to a vector is just under 7kb large. 

BR

Hi Sanna,

a few questions for you:

1) are you using random hex, oligo(dT) or gene specific primer for reverse transcription ?

2) did you try to amplify other (smaller) cDNA targets to check if RT is properly working ?

3) Amplification of a 7kb region is often challenging. Problems may occur not only in the RT but also during the PCR step. Which Polymerase are you using to amplify your target ? is the target GC-rich ?

4) Did you already try a nested PCR approach ?

5) Do you know the expression level of your target gene ?

Hi

1) I will use oligo(dT) to make sure I get a positive selection for longer fragments?

2) I have also ordered primers for a smaller part of my gene. But Im just about to start so no PCR has been preformed... I just wanted to make sure that there are no bad experiences with the Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit.

3) GC is 47% but Im just starting to do the RT. Im using long range PCR kit from Qiagen. 

4) No.

5) No. 

Thank you.

FYI

Amplifying a 6kb large fragment with cDNA synthesis with Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit  with  600 ng HeLa total RNA as input worked very well! :)

Thanks to everyone for there help. 

Sanna