PCR kit - (Aug/16/2011 )
Hi all,
I'm going to make order for PCR kit, But there are a lot of types,
Can you please help me by give me your suggestion which one to chose?
Thank you in advance
Noor
Hi noor,
It all depends on what your use will be. Are you going to do regular DNA amplification for cloning or? Some more details about your intended experiments will be helpful for a precise suggestion.
Cheers
Ho
prodes on Tue Aug 16 18:48:59 2011 said:
Hi noor,
It all depends on what your use will be. Are you going to do regular DNA amplification for cloning or? Some more details about your intended experiments will be helpful for a precise suggestion.
Cheers
That's right, thank you for remind me,
I'm going to amplify full length cdna, the gene size is about 3kb.. and the quality of cdna sample not very good because there are contamination with gDNA, as well as the primer not very specific, some PCR kit produce multi bands where others produce single band...
I personally was assembling the components I would need. In case of DNA amplification for cloning purposes I use high-fidelity proofreading DNA polymerases like Pfu, KOD, Pwo or some enhanced mixes like Herculase. Than, you need just any suppliers regular dNTP mix and well designed primers. In such a case I see no need to invest in a product sold as a "PCR kit".
What is the source of your template? You say that it is a cDNA contaminated with genomic. Did you buy a cDNA clone or you generated it yourself? If your target cDNA is in a plasmid you would be better off amplifying the plasmid first and minipreping it so that you get rid of genomic DNA. If you do not have such a choice you have to have rather long primers with Tm probably above 75degC in order to get the desired specificity. Finally, if such a strategy doesn't help to obtain a single band on a gel you could extract the desired band from the gel and reamplify it - in case your primers are correctly designed and you do not have repeats in the gene you are trying to clone you should obtain a single band.
Cheers
prodes on Wed Aug 17 13:40:08 2011 said:
I personally was assembling the components I would need. In case of DNA amplification for cloning purposes I use high-fidelity proofreading DNA polymerases like Pfu, KOD, Pwo or some enhanced mixes like Herculase. Than, you need just any suppliers regular dNTP mix and well designed primers. In such a case I see no need to invest in a product sold as a "PCR kit".
What is the source of your template? You say that it is a cDNA contaminated with genomic. Did you buy a cDNA clone or you generated it yourself? If your target cDNA is in a plasmid you would be better off amplifying the plasmid first and minipreping it so that you get rid of genomic DNA. If you do not have such a choice you have to have rather long primers with Tm probably above 75degC in order to get the desired specificity. Finally, if such a strategy doesn't help to obtain a single band on a gel you could extract the desired band from the gel and reamplify it - in case your primers are correctly designed and you do not have repeats in the gene you are trying to clone you should obtain a single band.
Cheers
The source of this gene from plant, and i used DNAse but still some gDNA contamination, also i try to purify single band from agarose gel, but after purification i didn't get result..
how can i design primer with Tm above 75'c?
I have a better grasp of your problem now. My first question is do you purify poly(A)+mRNA? I cannot recommend you a particular kit for the isolation of the template but a crucial step for the generation of cDNA is to have a highly pure mRNA sample without genomic for the RT-PCR step. Then, the RT-PCR you could do with generic dT primer or with gene specific. I would rather go for a long, gene specific primer. It appears that the AMV RT would do good job for the RT-PCR and then you can use Taq or high-fidelity DNA polymerase to generate the cDNA.
The Tm of a primer depends on its sequence and length. Check this site for more information: http://www.promega.com/techserv/tools/biomath/calc11.htm#disc
prodes on Sat Aug 20 16:51:17 2011 said:
I have a better grasp of your problem now. My first question is do you purify poly(A)+mRNA? I cannot recommend you a particular kit for the isolation of the template but a crucial step for the generation of cDNA is to have a highly pure mRNA sample without genomic for the RT-PCR step. Then, the RT-PCR you could do with generic dT primer or with gene specific. I would rather go for a long, gene specific primer. It appears that the AMV RT would do good job for the RT-PCR and then you can use Taq or high-fidelity DNA polymerase to generate the cDNA.
The Tm of a primer depends on its sequence and length. Check this site for more information: http://www.promega.c...calc11.htm#disc
Hi,
I didnt purify mRNA, and for gene specific primer i used set of primers from article, but some was working and some didnt work, i'm confused why, not sure whether the problem from my template or may be because of the primer is not specific, do you think the DMSO may solve the problem of specificity of the gene?
And what is the AMV RT, is it kit brand?
Thank you v .much for your suggestion
I tested a lot and the best is Clontech's Advatage GC Genomic LA