4 replies to this topic

[sen111]

Hello Everyone,
                         I am trying to clone 4.5Kb gene from total RNA isolated from Placenta. I reverse transcribed total RNA into cDNA using Roche Transcriptor RT kit (has RNase H activity). Then I did a PCR with my primers specific for my gene of interest. I didn't get any amplification (My primers & PCR conditions were fine). Is it necessay to do second strand cDNA synthesis before doing PCR?

If so can i use Klenow fragment(Fermentas) for second strand synthesis? I came across lot of protocols using E.coli DNA polymerase 1, RNase H & E.coli DNA ligase. I am looking for a protocol using Klenow.


Thanks in Advance
Sen111

[almost a doctor]

Hi Sen111,

when you say:  "My primers & PCR conditions were fine"   what do you mean exactly?  Do you have a positive control?  I mean, if you didn't get any amplification most likely some of your PCR might not be fine, be the primers, the conditions, the reagents, the template....  
I don't think you need to do second strand cDNA synthesis (I've never bother with this and I've always obtained amplification from my cDNA), more likely you'll need to repeat your RT and optimise your PCR. If you are 100% certain your PCR is fine (ie. you have a positive control that works) maybe you had too much/too little template or simply your RT reaction didn't work.

[sen111]

almost a doctor, on 31 August 2011 - 06:24 AM, said:

Hi Sen111,

when you say:  "My primers & PCR conditions were fine"   what do you mean exactly?  Do you have a positive control?  I mean, if you didn't get any amplification most likely some of your PCR might not be fine, be the primers, the conditions, the reagents, the template....  
I don't think you need to do second strand cDNA synthesis (I've never bother with this and I've always obtained amplification from my cDNA), more likely you'll need to repeat your RT and optimise your PCR. If you are 100% certain your PCR is fine (ie. you have a positive control that works) maybe you had too much/too little template or simply your RT reaction didn't work.

Hello Almost a Doctor,
                                  Thanks for your reply. Yes i used positive control (Same gene (but a point mutation in the middle) cloned in a plasmid). It worked fine. Ok i will increase the cDNA template & try it again.

[noyara]

Hi Sen111
your project very similar to mine,

may i ask you why you looking for clone your gene? do you want to express to protein?
and can you please tell me which cloning kit you will use?

i try to design primer to get full length cDNA about 3kb and cloned into expression vector, but so confused which one to chose..

can you please suggest what is the suitable one...

Thank you v.much in advance
Noor

Edited by noyara, 14 September 2011 - 07:35 AM.

[sen111]

noyara, on 14 September 2011 - 07:33 AM, said:

Hi Sen111
your project very similar to mine,

may i ask you why you looking for clone your gene? do you want to express to protein?
and can you please tell me which cloning kit you will use?

i try to design primer to get full length cDNA about 3kb and cloned into expression vector, but so confused which one to chose..

can you please suggest what is the suitable one...

Thank you v.much in advance
Noor

Hi Noyara,
My PCR worked after i did second strand cDNA synthesis (using Klenow). I am yet to sequence it but i got product of expected size. I didn't use any kit. I used phusion high fidelity dna polymerase from NEB. Roche transcriptor cDNA synthesis kit (1st strand). T4 ligase from NEB.

You can choose any product. I am happy with Phusion enzyme, i could able to amplify 4.5 kb gene.

Yes i want to express this in cell lines.

Regards,
Sen111

Edited by sen111, 19 September 2011 - 12:18 PM.