Hi

Whats the taq you have been using? any thing above 1kb amplicon size you have to use high fidelity taq. you may take a look at this it may be helpfull for you.

http://tools.invitrogen.com/content/sfs/manuals/cms_041556.pdf

Regards

Jam

I believe, since your product size is too huge, you should be be trying nested PCR. I mean, amplify your target gene cluster with a primer for the out range which can cover your target gene. When you will get the proudct from the first amplification it will be shorter and now this product will acts as a starting material (template) for the second PCR. While running PCR, use the exact PCR primer for that gene which can exactly amplify that (your target) gene.

I think, you should read about the nested PCR first before doing it. Usually, nested PCR is done for the target gene which is too huge in size.

This PCR is little bit cumbersome but I still believe it is worth doing it.

Just try it. Hope you wil get your desired result.

Wish you all the best for your work ahead.

With warm regards,

Rajesh Chaudhary

Hi Graca

The fragment size of 2400 bp should be possible to amplify using any routinely used enzyme for PCR amplification...What type of problem you are getting......if you are not getting any product or no amplification there can be several reasons for it....I suggest you also do a positive control along with your gene of interest....by positive control I mean any fragment of DNA which has been working always when you do a PCR....if you don't have such a fragment...inquire with other researchers in the lab.....this will indicate if every component in PCR is working fine or not

bye

Hi Ajay!

Thanks for your attention.

I used High Fidelity (invitrogen) and I tested ten temperatures and 3 different Magnesium conditions and I don´t have any product. I´ve used a housekeeping and the PCR worked. So I think I have to use another enzyme, more specific, but I don´t know what.

Thanks

Hi Krzysztof Wicher! I´ve tested 60 different conditions and I can´t have success in PCR. I think ir´s the enzyme, I need one more specific, I don´t know. Thanks.

Hi Jamuna! I´ve used High Fidelity (Invitrogen) but it didn´t result! I will see your options! Thanks!

Hi Rajesh! I will study Nested PCR and maybe I will try it.If I have another problems I will tell you! Thanks for your answer!

Hello Graca,

Before you go for the nested PCR. But, before that I would like to suggest you few things. I am not sure, but I guess you might have done these things or if not, then make sure of few things. Such as, check the concentration of your template DNA you are trying to amplify your gene of interest and make sure it doesn't have any kind of contamination including protein or enzymatic contamination that might have persist while doing DNA extraction process.

Secondly, try to run GEL ELECTROPHORESIS and check whether even if you get some kind of smear or not. If there is something like smear, change your primer concentration. Too high concentration of primer might inhibit reaction. Make sure of your annealing temperature which is more crucial in PCR and the enzyme activation step. Make sure your enzyme has activated to its higher possible level so that it can function properly. Sometimes our DNA doesn't get seperated, thus it will ultimately not show any product. If you doubt about your DNA that it might not be getting seperated or uncoiled -- try using DMSO concentration between 5 to 10% concentration -- starting from 5% concentration. But, do not use DMSO if you think that your DNA lineralize quite well with your induced thermal profile, because DMSO might inhibit your reaction as it has got negative effect on Taq DNA polymerase -- it shortens half-life of Taq Polymerase.

What is the size of your primer? Run BLAST with your primer sequence and try to make sure about its melting temperature.

I think, you might have done these troubleshooting, but if not then please do it. It might help you someway, I guess.

Wish you all the best with your work.

Have a great time ahead.

With warm regards,

Rajesh

Hello Graca,

can you test your enzyme with a PCR reaction which is known to work in the past? That would be a way to test if the enzyme works. In my experience its usually the enzyme that fail, since taq polymerases are pretty stable.

Do you know your primers are working? Can you exclude the possibility that you made a mistake while designing them (just listing different possibilities)? Are you sure that one of your primers binds to the reverse strand?

How does your gels look like, do you get a smear? Do you get primer dimer bands or are they simply empty?

best regards

Christian

Hi Graca

I have a query.......are you trying to amplify the gene (2400 bp) from genomic DNA or cDNA? since you are taking about 'housekeeping gene' I GUESS you may be trying to amplify from the cDNA.....if this is the case

1) You need to check your RNA quality...and also try to assess the quality of cDNA synthesis as 2400bp gene is easy to amplify from the genomic DNA (provided the quality is good and is not sheared)....such a long gene will be difficult to amplify from the cDNA.....(in this case the RNA quality and cDNA quality needs to be good)

2) In case of cDNA...you should try specific Reverse Transcriptase..which synthesis long cDNA molecules...such as 'Expand Reverse Transcriptase' from 'Roche' or simlar products from the other companies......but they will also synthesize long cDNA (of longer genese ofcourse) if the RNA integrity is good...

3) If it is cDNA as I guessed...then small fragments of housekeeping gene or other genes (including your gene) may be possible to amplify...but amplifying long fragments will be difficult ...and would be possible only if abovementioned points are taken into account

4) One more possibility ...is if you are doing total cDNA synthesis using Oligo-dT primer...and using it for housekeeping gene and gene of interest....for the later I'll suggest you do RT using gene specific primer and see the results...

I hope I am guessing the right points and the my suggestions may be of help to you

2400bp should definitely work but I would need to know your reaction conditions, specifics to be able to suggest something

hi

i think what Susan and Victoria has mentioned is absolutely right. please let us know the PCR conditions along with primer details.

Regards

Jamuna