Anwar- this may simply be a function of too many cycles or overloading your gel. As long as your PCR product is specific (not occurring in negative control E. coli), then you don't need to troubleshoot. A band, smeary or not, can be indicative of a positive clone, which you will subsequently verify by sequencing. Colony PCR is inherently a quick and dirty screening method, so perhaps not worth time optimizing to perfection.

The critical question is not how sharp the band is, but whether it is a true positive signal for recombinant plasmid.

A common problem is too many cells in the PCR reaction. Try diluting your cell suspension by 10x and 100x.

Upward smears, beginning at the band area and stretching up toward the wells on agarose gel, are indicative of RNA contamination. Reducing the amount of DNA added or diluting it is a quick way to eliminate it, could also do RNAse treatment.

Regarding other causes of smear in PCR, these could be:

Using too much DNA template;

Using too much Taq polymerase;

Magnesium concentration is not optimal;

Too many PCR cycles

Non-optimal Mg++ concentration

Suggestion

Titrate magnesium concentration using our PCR Optimization Kit.

Nucleotide concentration is too high or unbalanced

The standard concentration is 20 - 200 µM of each nucleotide.

Suggestions

Check the concentration of stock solutions of all nucleotides

Double check the final concentrations of all nucleotides

DNA contamination / carry-over

Suggestions

Test for carry-over by performing PCR without adding target DNA

Avoid carry-over (see below)

To prevent carry-over, good lab practices should be used such as:

Physically isolating PCR preps and products

Autoclaving solutions, tips, and tubes

Aliquoting reagents to minimize repeated sampling (no more than 20 reactions per aliquot)

Eliminating aerosols by using positive displacement pipettes

Premixing reagents

Adding DNA to reaction last

Choosing positive and negative controls carefully

Soaking gel box and combs in 1M HCl to depurinate DNA

Using new razor blades to excise bands

Covering UV box with fresh plastic wrap

Always using oil overlay

To eliminate contamination / carry-over:

UV irradiation: Mix all components, except template DNA, irradiate in clear 0.5 ml polypropylene tubes in direct contact with glass transilluminator (254 and 300 nm UV bulbs) for 5 minutes

UNG Digestion: Incorporate d-UTP nucleotides into reaction and do subsequent uracyl DNA glycosylase digestion

Primer annealing temperature is too low

Primer annealing temperature is typically 50 - 60°C (may be higher or lower based on primer sequence and buffer components).

Suggestion

Determine Tm / annealing temperature with our convenient Tm Calc benchmate based on the following equations:

If primers are 20-35 bases

Tp = 22 + 1.46(Ln)

Ln = 2(# G or C) + (# A or T)

Tp = Effective annealing temperature ± 2 - 5

If primers are 14 - 70 bases

Tm = 81.5 + 16.6 (log10 [J+]) + 0.41 (% G + C) - (600/l) - 0.063 (% Formamide) + 3 to 12

[J+] = concentration of monovalent cations

l = length of oligo

Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers

Suggestions

Increase initial denaturation temperature to 95 - 97°C

Denature DNA minus enzyme and buffer for 4 - 6 minutes

Increase cycling denaturation time 15 - 30 seconds

Try "Hot Start" technique

Add T4 Gene 32 protein 3 - 5 µl/ml

When designing primers, make sure there is no more than 2 bases of homology at the 3' end. Use a primer design program if available.

Consider addition of cosolvent to reaction buffer:

3 - 15% DMSO

1 - 10% Formamide

5 - 15% Polyethylene glycol

10 - 15% glycerol

DNase activity (indicated by smears visible on gel below expected band size)

Suggestions

Make fresh stock solutions

Check the integrity of template DNA

Oil contamination of gel sample

Suggestion

Spin the reaction tube and carefully extract the oil layer from the surface

Incorrect template to enzyme ratio

The necessary amount of template varies from reaction to reaction. As a guideline, 100 - 750 ng human DNA (105 - 106 copies) per 100 µl reaction. The amount of enzyme should be optimized for each template.

Suggestions

Titrate the amount of template in the reaction

Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units)

1) Dilution series of template

2) Reduce number of cycles

3) Use new reagents and clean pipettes

4) Try suggestions for multiple bands >50 bp

5) Lower enzyme concentration

6) Optimize MgCL2 concentration

7) Optimize primer concentration or use fresh primers

8) Optimize primer design

You can also read this article from Cold Spring Harbor Protocols regarding to troubleshoot the problems of pcr including colony pcr product smearing as attached below;-

Also, check this link for getting know more about the discussion on colony pcr:-

https://www.researchgate.net/post/Protocol_for_colony_PCR

Hi Anwar

The most likely reason is too many cells in the PCR reaction..which can happen if your using a regular loop for picking colonies for PCR.....we generally use a very fine needle to 'stab' the colony and put it in the PCR mix.....this does not even disturb the colony and just picks the sufficient number of cells for a very clean colony PCR ..most of the times.....You may also have to take into account the copy number of the plasmid you are using for genetic engineering experiments

bye

Anwar- this may simply be a function of too many cycles or overloading your gel. As long as your PCR product is specific (not occurring in negative control E. coli), then you don't need to troubleshoot. A band, smeary or not, can be indicative of a positive clone, which you will subsequently verify by sequencing. Colony PCR is inherently a quick and dirty screening method, so perhaps not worth time optimizing to perfection.

The critical question is not how sharp the band is, but whether it is a true positive signal for recombinant plasmid.

As has been said, likely too much DNA was present. We pick the colony, streak it on a new plate as to keep that colony for use afterwards if your PCR works, then dip that in your reaction.

Have you ever tried to add a final extension step?

Sometimes, when you are amplifying with a short extension into the cycling, the taq is not able of copying the whole fragment.

If you add a final step at 72°C/ 5' (or the optimum temperature for your taq) you could get a good band.

Try colony PCR with lesser amount of colony/DNA, lesser amplification cycles and a final extension of about 3-5min