RAPD Randomly Amplified Polymormphic DNA PCR

[admin]

Hello everyone,
just received a message from a user with this question:

I have been working on RAPD (Randomly Amplified Polymormphic DNA).

Im working under the DBT project as finding the molecular markers for growth in Fish.
I was getting good results with Peqlab Primus 98 thermocycler machine with the following concentrations:
DNA 15ng= 2 micro litre
dNTP 5mM=1 micro litre
taq bufferA10x = 2 micro litre(which comes along with the Taq polymerase from the company GENIE)
Taq polymerase (GENIE)3u/microlitre= 0.4micro litre
MgCl2 (25mM)= 1.2 micro litre primers screened and using (SIGMA COMPANY)2pM=12micro litre
OPG 9 OPP16 OPB17 OPA7 OPF9 OPG4 OPF 10

But now suddenly i have started getting smear of DNA.
Ive tried with different concentrations of DNA, MgCl2, Taq. But still I'm not getting results. Can you please help me out in solving this problem.

thanks guys

[megha]

u can increase annealing temp upto 39 C, or ckeck your stocks of dNTPs, taq, buffer, n MgCl2 by taking negative control,,,u should check if ur stocks r really stored in -20/-80 C.
megha

[K. ASHOKKUMAR]

use proof reading TAQ polymerase (PR Polymerase) with mgcl2
use 5ng DNA for PCR reaction.

[samuel moses]

have u checked the quality of the DNA you have used for amplification, i think the problem is with the quality of the DNA, that is all the problem

Originally Posted by admin

Hello everyone,
just received a message from a user with this question:

I have been working on RAPD (Randomly Amplified Polymormphic DNA).

Im working under the DBT project as finding the molecular markers for growth in Fish.
I was getting good results with Peqlab Primus 98 thermocycler machine with the following concentrations:
DNA 15ng= 2 micro litre
dNTP 5mM=1 micro litre
taq bufferA10x = 2 micro litre(which comes along with the Taq polymerase from the company GENIE)
Taq polymerase (GENIE)3u/microlitre= 0.4micro litre
MgCl2 (25mM)= 1.2 micro litre primers screened and using (SIGMA COMPANY)2pM=12micro litre
OPG 9 OPP16 OPB17 OPA7 OPF9 OPG4 OPF 10

But now suddenly i have started getting smear of DNA.
Ive tried with different concentrations of DNA, MgCl2, Taq. But still I'm not getting results. Can you please help me out in solving this problem.

thanks guys

[samuel moses]

have u checked the quality of the DNA you have used for amplication, i think the problem is with the quality of the DNA, that is all the problem, so if

[ur99.pawar]

check the DNA concentration, are u sure that u are using exactly 15 ng DNA. if DNA quantity is more you will get Smeared product. probably that,s problem.