I have trouble in amplifying a gene. I am continuously trying to find a possible solution. All methods have failed me somehow. I was amplifying gene A using gene specific primers. This gene has a size of 1000 bp, with intron and was amplifying correctly (using genomic DNA), when I used biotools polymerase. But, now when I am trying to amplify it with a different polymerase (thermoscientific poly with KCL buffer and MgCl2 suppied separately) at same annealing temperature, I do not get any amplification. Recently, I thought of amplifying it with cDNA. I used a control gene B that has been cloned for setting up a PCR. The experiment is as follows:
On gel (attached picture, from left to right)
Ladder-100bp
Control
Gene B- with cDNA
Gene A - with cDNA
Gene B- with genomic DNA
Gene A with genemic DNA
I am not getting any band for this specific gene A. Help?
Hi Sakshi,
Have you used the same gDNA sample for gene A and gene B amplification? If that is the case, you have to optimize your PCR conditions. If not, then I would try to get a cleaner gDNA sample for your gene A.
I would recommend you to use DMSO in your PCR reaction as it helps to denature your complex template (gDNA) and to screen different temperatures with the thermocycler making a gradient. Hope it helps.
@Xi Xang I Have used same DNA sample for the amplification of the genes as they are two genes from a single plants which needs to be amplified. Morever, I varied PCR condition, nothing is working. But earlier I got bands for the same gene with another polymerase now I am just not getting the results.
I would agree with Xi Jiang, try add DMSO to increase amplification specificity, and perform a griedient PCR to find out the best annealing temperature.
I have already set up a PCR gradient earliers which showed max. Amplification at the temperatures which I used this tome with another polymerase. DMSO is already added in the reaction as my gene is GC rich
The ingredients in the 10x buffer is a little bit different from other ones (see below). Why they add Nonidet P40 ? What is the function is that? Do you have other set of polymerase kit for PCR in your lab?
10X Taq Buffer with KCl and 15 mM MgCl2 (B16) includes:
• 100 mM Tris-HCl (pH 8.8 at 25°C)
• 500 mM KCl
• 0.8% (v/v) Nonidet P40
• 15 mM MgCl2
https://www.thermofisher.com/order/catalog/product/B16
Very good point Yuan-Yes Yau, in the link you may find attached, it says that non ionic detergents prevents secondary structures of the template and stabilizes the polymerase but it may increase non-specfic amplification. I think Sakshi can try making the buffer without the NP40 and try again the PCR :)
https://www.genelink.com/Literature/ps/M40-3021-PCR_Additives_Ver5.1.pdf
Thanks for the attachment @Xi Jiang. If the 10X buffer already contains this PCR addtive--Nonidet P40, adding extra addtive, such as DMSO, might screw up some of the PCR amplification.
By the way, @ Sakshi Mehta, in your picture, the PCR product sizes from either cDNA or genomic DNA of gene B seem similar. What is the size of 'introns' in gene B?
The gene has no introns and hence the size is same. I was just using it as a control for both genomic as well as cDNA amplification to check what is actually wrong with the polymerase.
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