Hi,
I am new to research and having a difficult time understanding the meaning behind some things. I am working on an experimental PCR where we are doing SDM and mutating a plasmid with mutagenic primers. Then, we transform E. Coli with the mutant plasmid, and then purify plasmid and verify mutagenesis. I understand all this part. Alongside, a smaller project is creating a control PCR reaction with a given control plasmid for SDM. We are instructed to run a gel after creating the control PCR to see if the primers bind. My question is, what exactly do the bands mean on my gel of my control PCR?? My bands were very faint but regardless I am confused. The control plasmid is 4.5 kb. Where is the band expected to show compared to a ladder?
Please mention how you are planning to design your control? Is it a negative control, or a positive control?
If you are doing a PCR, the desired segment between your primers will get amplified. Use Snapgene or similar software to find out the length of your PCR product (in Snapgene you can keep track of all of your constructs, along with primers and other details).
If you have a positive control, you will get a band; but if you have a negative control, there will be no bands. The position of your band will be next to the similar-sized band on the ladder. To know the size of the bands on the ladder, please download the datasheet from the manufacturer's website.
Biswajit Biswas Ok so are you saying that what is amplified is the primers sequence? Also I believe the control PCR is positive... We created a master mix (50 ul), and then separated them into 2x25 ul PCR reactions - 1 had 24.5 ul master mix and 0.5 DNA pol and the other had 24.5 ul master mix and 0.5 ul H2O. What is the purpose of adding DNA pol to one and H2O to the other? Bands showed up in both lanes, with the H2O PCR lane slightly lower than the lane with DNA pol.
If you are doing gene mutation ,and that particular gene is successfully mutated,then on gel u didn't get your expected band, where as in control you get your expected band.
To find the weight of your expected band please use snapgene tool.
Sounds like you need to sit down and have a chat with your research mentor. The purpose of having one reaction with water instead of DNA is to set up a "negative control" to check your reactions for contamination. The lower band is (hopefully) just unincorporated primers from your PCR reaction (aka "primer dimers").
PCR is a really common tool in molecular biology and you'll need to understand how it's used to really understand your project.
Good luck!
Using PCR, a DNA sequence can be amplified millions or billions of times, producing enough DNA copies to be analyzed using other techniques. For instance, the DNA may be visualized by gel electrophoresis, sent for sequencing, or digested with restriction enzymes and cloned into a plasmid.
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