4 replies to this topic

[hercolanium]

I am working on vitamin D receptor (VDR) gene, and my primers are:

Taq I: 5’-CCTTCTTCTCTATCCCCGTG-3’ (Forward) and 5’-GCAGGTCGGCTAGCTTCT-3’ (Reverse)

And

Fok I: 5’-ACTCTGGCTCTGACCGTG-3’ (Forward) and 5’-TCATAGCATTGAAGTGAAAGC-3’

I want to amplify the gene by PCR; I have tried many protocols and no band appears even though when I run the samples before the PCR I get clear bands

I am using Promega master mix 12.5 ul + 1ul reverse primer + 1ul forward primer + 10 ul DNA sample + 0.5 ul nuclease free waterà 25ul finishing solution

Used protocols:

Initial denaturation at 94° C for 5 min, 35 cycles at 94° C for 30 s, 58° C for 30 s and 72° C for 60 s and one final cycle of extension at 72° C for 7 min…

I have changed Initial denaturation to 95° C once; also I have increased the annealing temperature to 59° C.

I have increased the primers in place of the nuclease free water and I have increased the DNA sample

 

What can I do to perfect my protocol?

[hobglobin]

Did you checked the DNA concentration before? I work usually with 1-2 microL per  reaction with maximum 50 ng. Too much DNA can also disturb the PCR.

[hercolanium]

Did you checked the DNA concentration before? I work usually with 1-2 microL per  reaction with maximum 50 ng. Too much DNA can also disturb the PCR.

How can I make sure that I have the right amount? I usually run my sample in gel electrophoresis (5 ul from purified DNA tube + 5ul loading dye) and the band comes out very clear

[phage434]

I agree with Hobgoblin, you likely have vastly too much DNA template. Dilute 10x and use 1 ul of that template in your reaction. Also, what is the concentration of your primers? What is "finishing solution?"

I would try lowering the annealing temperature to 52-55. How long a product do you expect?

[hercolanium]

I agree with Hobgoblin, you likely have vastly too much DNA template. Dilute 10x and use 1 ul of that template in your reaction. Also, what is the concentration of your primers? What is "finishing solution?"

I would try lowering the annealing temperature to 52-55. How long a product do you expect?

 

Should I dilute the DNA with nuclease free water or with extra DNA rehydration solution from Promega kit? I use 1 ul forward + 1 ul reverse primers which I have diluted according to the manufacturer leaflet then diluted them further into a stock tube then more into a working tube (I will bring the detailed concentrations soon when I go to the lab) my finishing volume is 25 ul including the primers, sample, master mix and 0.5 ul nuclease free water

 

The expected products are:

 

TaqI

T(T) allele: 174 bp

C(t) allele: 58 bp + 116 bp

->TC(Tt): 174 bp+58bp+116bp

 

FokI

 C(F) allele: 159 bp

T(f) allele: 53 bp + 106 bp

->CT (Ff): 159 bp+53 bp+106 bp