The PCR reaction worked well before and i got clear sharp band of my desired length. But now the PCR is resulting to smeared product with no band of the desired length. i have newly isolated the genomic DNA, diluted the template and primers and still the problem is same. please suggest me on how to solve this
Your problem can be sorted out step by step.
Negative control-please see if getting band in negative also. Band should not be coming in negative control. It indicates contamination..
MgCl2 concentration
Hot start PCR conditions and master mix can be used
long PCR cycle may be avoided.
Try to use BSA, formamide, touchdown phase in the PCR, not correct Tm annealing (I suggest to test a gradient).
How much DNA did you use? How is the origin of the DNA? Because is possibile that something solvent from the DNA extraction interfere on the PCR.
The following advices were given by QIAGEN.com
Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:
- too much starting template Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
- carry-over contamination If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
- enzyme concentration too high When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
- too many PCR cycles Reduce the number of cycles in steps of 3 cycles.
- Mg2+ concentration not optimal Perform PCR with different final concentrations of Mg2+ from 1.5–5.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see table below):
Final Mg2+ concentration in reaction (mM)1.52.02.53.03.54.04.55.0 Required volume of 25 mM MgCl2 per reaction (ul)02468101214
- Primer concentration not optimal or primers degraded Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.
- Primer design not optimal Review your primer design, and design new primers.
At first, you should try a gradient PCR to optimize the annealing temperature of the primer that you are using.
Dear Tanvir,
Check/do few things:
1) Quality and quantity of your DNA;
2) try a gradient PCR;
3) adjust MgCl in the PCR;
4) consider a negative control to look for possible contamination.
Best of luck.
I agree with Basanti Jyotsana that post pcr contamination is the likely reason and it is essential to run a no template control and if this produces a pcr band then you have contamination and this will be a serious problem
I have tried every possible troubleshoot to solve the problem but the result is same.
1. Worked with diluting the initial primer and template to lower concentration but the result is same with fade smear.
2. The primer annealed previously resulting sharp band at 48oC. as it is not giving any band now, i have tried 46, 50, 52 temperature but no result.
3. As we work using ready made master mix, we cant adjust the MgCl concentration
4. No template PCR does not produce any band.
From this outcomes, any suggestion to solve this?
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