The plasmid that was transformed originally is correct (sequenced). I am expressing different gene targets from the yeast genome to optimize my protein. When I sequence the incorrect size, it matches one of my gene targets (also from the yeast genome) , not the one I transformed and it happened with many targets (they all match with one of the gene targets from the genome so this is not an operational mistake. My colony PCR Primers are on the promoter and the terminator and I check all the targets with the same primers. I got many right sizes too (targets from the yeast genome) in the same batch of transformation.
I have change primers and the Tms too, but the gel is either empty or the sequence matches with another target not the one I transformed.
I am not sure I indersyand what happened.
Is the incorrect target shorter than correct target ? PCR reaction preferentially amplifies shorter targets, if there are more options.
Did you do a lot of PCR with the incorrect target before you started have these problems ? PCR products can sometimes get into aerosols and contaminate next PCR.
Did you run a BLAST and check if your primers have any other homologies in yeast genome? Colony PCR is tricky. The verification primers are shorter in size and they tend to bind non-specifically. Sometimes colony PCR results in multiple non-specific bands with the specific band. You can try to increase the annealing temperature 1-3 °C to enhance the specificity and reduce the extension time to omit non-specific products with higher molecular weight. Also, keep in mind that recombination can occur non-specifically during transformation.
Aqsa Ahmed hello, i agree with Barbara Brezna as i also did not understand your question completely. Moreover, as much as i can understand, you are saying that when you checked your transformed vector via sequencing you get the correct target gene. However, in colony PCR the size is different but sequencing it revealed that it contains the sequence of your target gene. Now, there can be two possibilities
1. your plasmid contains the target gene but your insert (Target) is bigger than you are expecting.
2. Your colony PCR primers are amplifying a region which is bigger than your target gene.
It will be very helpful if you try and explain your question a bit more.
Thankyou
Thank you everyone. I will try to explain more.
First of all, my all gene targets (correct or incorrect) are all from the yeast genome.
They all vary in sizes.
I transformed 18 gene targets in total (one by one).
I got 6 right according yeast colony PCR (correct band, correct sequence).
But I also got single bands on the gel for 5 other gene targets having the same size (incorrect size).
When sequenced, they matched with one of my gene targets but I am sure I did not transform that gene target 5 times.
It happened in the same batch of transformation.
I will of course repeat the transformation but I was curious why did it happen.
If you still have ambiguities, Your further questions are welcome.
Barbara Brezna Thank you for your response.
The incorrect gene target is not smaller than the correct one.
Also, can you please elaborate the PCR contamination issue?
Satyendra Mondal Thank you for your suggestions. Increasing the annealing temperature gives me either an empty gel or multiple bands. I got a single band only at tm 51, that I have been using for my primers on the promoter and the terminator. I am aware that there can be an unspecific recombination but my curiosity lies in the fact that it matches that one gene target in the genome. BLAST sounds good, I will definitely do that.