Dear V Phung,

Sometimes handling issues cause primer dimmer and other non-specific bindings. It also may happen that in your primmer.

Change your primer with other vendors this may help you.

It's may be your primer problem- it may bind nonspecifically or make primer dimer. Try to change primer or modify annealing conditions!

It may be due to the nonspecific binding of the primer to the gene of interest. If the primer sequence and the sequence of the gene of interest is known to you, then you can run a primer BLAST to check whether the primer is binding at multiple position of the gene.

One of the possible reasons for multiple bands is contamination of the DNA.

Van Phung

There are some reasons to false band in PCR products

1. High concentration of primers

2. Template DNA contaminated with RNA.

Hi,

Please don't jump into any conclusion without some background data.

First of all, you need to verify that the transformation assay worked well or not. Without confirmation of successful gene transformation , there is no way to optimize or troubleshoot PCR results. I hope, you might have some phenotypical observation to confirm that the transformation happened successfully. If not, you need to do PCR with template extracted from before and after transformation. Extract DNA before and after transformation assay. If the PCR protocol is well established or published, run in parallel. If the primers are new or designed by you, then stick to standard concentrations.

If possible share some additional information like master mix, primer concentration, template concentration etc used in the previous assays.

all the best

Kalyanaraman

One of the likely causes of multiple bands in PCR is nonspecific primer annealing. ... Too many PCR cycles (more than 30) also has the potential to cause multiple bands due to the increased chance of error with each cycle. DNA contamination is another possible factor. Van Phung