Whats your electrophoresis conditions? TAE or TBE?

Try to prepare fresh buffer and run gel in low voltage?

Are you using low EEO agarose?

Hi

Dear Dhanasekaran Sakthivel

I used TBE buffer , Fresh buffer and voltage 80.

Thanks your comment.

u can try TAE buffer that is good one..but the buffering capacity is less compare to TBE..so u have to change the buffer frequently..i feel some problem in ur apparatus..smearing may also be due to bad quality DNA sample..see your marker it does not have any smear..try to use TAE with 45-50 V and if still the center wells move slower than end ones change the apparatus and try again..hope u will succeed...

I would suggest you to consider the following parameters

1) Check the pH of your buffer.

2) Try running the electrophoresis in cold condition.

3) Is your electrophoresis apparatus electrodes are ok? make sure that it is straight and not corroded. hopefully this may help.

Lets try. Cheers

salam kako

1-first optimized your PCR reaction to give single and clean band ( Tm or Mgcl2 gradiant)

2-if DNA as template you could change your DNA extraction methods to achive poure and clean DNA.

3-If you used cDNA as template , you could 1ul cDNA+2ul DDW = 1 ul as template

4-You could direct PCR from yeast ( 1 colony from yeast diluted in 10ul DDW= 1 ul as template )

5- you could decreas concentration of your primres( insted of 1ul from 10 mM , 0.5 ul uptake).

6-Finally high concentration of template or Impurities could be main reason for smear in PCR reaction.( OD260/280 must be 1.8 -2 )

If your problem is uneven migraton between the middle lane and the lanes on the side, there are 2 possible causes/solutions.

1. Curved electrode (on either side)--> solution: change the running chamber...look for stright one.

2. Uneven polymerization of the gel --> solution: melt the gel well, pour while it's still hot like ca. 2-3 min after melting (make sure you use good quality tray though...otherwise it may crack), and pour in quick, single movement fashion (pour all at once, and make it quick). With this trick, my gels always stright like a ruler ^ ^

Good luck.

You have a consistent uneven current across the gel. This is usually caused by edges of the gel being warmer (higher current) than the interior. Regardless of voltage, keep current below 50mA. Measure the thickness of your gel centrally and laterally to prove your mold is forming a uniform gel. Also, use 0.5XTBE and you decrease the current tw0-fold