I use PCR to confirm the insert in my clones. Normally its getting positive or negative bands. But this time I got bands less intense than the positive with primer dimers. some clones only got maximum intense band but no any primer dimers. I am confuse with this result as I cannot confirm the positive clones.
what are those less intense but same size bands produced by the PCR. Are they result of any Primer related artifact or a contamination.
Negative sample didn't get any bands other than the primer dimers.
I would not pay too much attention to the band intensity. Usually, you take a small part of a colony for colony PCR. However, this leads to very different template concentrations in your PCR mixture. It could thus be that the formation of primer dimers competes with binding of primers to the template DNA and depending on the template amount, one or the other situation is favoured. As long as you get a band with the expected size, I would think that the clone is positive.
Good luck!
I would not pay too much attention to the band intensity. Usually, you take a small part of a colony for colony PCR. However, this leads to very different template concentrations in your PCR mixture. It could thus be that the formation of primer dimers competes with binding of primers to the template DNA and depending on the template amount, one or the other situation is favoured. As long as you get a band with the expected size, I would think that the clone is positive.
Good luck!
the intensity of the band depends on the DNA concentration, in this case on the colony i.e. how much you get for PCR.... I agree with Alexandra Muller, you should avoid the intensity of bands, because colony PCR is just to see the positive clone. Further confirmation is usually made by sequencing, a common practice for identifying the right colony containing your gene of interest....
Good Luck
Hello Nilmini,
Like Alexandra mentioned, number of bacteria inoculated in a PCR tube vary greatly from tube to tube. Tubes that received too many or too few bacteria will give you weaker bands compared to those that received just the right number. Too many bacteria will potentially inhibit the PCR due to both excess of DNA template (can even be worse for high copy number plasmids) and PCR inhibitors. After doing practically thousands of cPCRs, I have learnt that less is good when it comes to how many bacteria should be added in the tube. There are some 1-2 x 109 bacteria in a single 1-2 mm colony. Typical inoculum on the tip of a toothpick can easily contain 107 or more bacteria, too high a number for a sensitive technique such as PCR. To circumvent excessive inoculum problem, I just touch the colony with a sterile toothpick and draw a cross on the patch plate. Next I dip the tip (with whatever bacteria left on it) in the PCR reaction. This delivers just the right number of bacteria in the PCR reaction and reproducibly yields almost similar intensity bands for positive clones.
FYI - LB Agar inhibits PCR. But I agree with all the above comments
Hello Nilmini
You can take it as positive as everyone have suggested. since you got the band at right size and no bands in negative control. The reason the less intense band is due to
1. Low DNA concentration
2. Annealing temperature
Though the melting temperature is constant for a primer. Annealing temperature for bacteria changes (even within same sp but different isolates). If your interested you can do a gradient PCR to figure that out.
i agree with concentration of DNA play a good role in PCR cycle but in the above question is why faint band and why bright band resion behind it which bacteria got proper insert show always good result as well as bright band which bacteria have less amount incert then its show band quality is very bad.
see the above mentioned reason are true but i will add some point here in colony pcr ur fragment size usually get increase then the normal pcr product size so u need to be judge ur insert precisely not only dat colony pcr also depend upon the efficiency of competent cells u are using to get it transformed with proper dna concentration.so change ur competent cells and i will advice to use XL1-blue competent cells.
secondly, when u r using etbr staining to check ur pcr product den dont add etbr in the agarose gel becoz wen u do this then the gel is not stained with equal proportion n u get some low band and some with high intense band. just u run the sample first in ur agarose gel then make etbr solution of 0.5microgram/ml in autoclaved distill water and then put ur gel in dat solution and left it for 1-1:30 hr in shaking mode u will see dat ur staining is in equal proportion and u will not get any low or very intense band.
Hi there,
To be sure about the significance of the low intensity band you may get I suggest you to always run two PCR negative controls : one with an plasmidless clone and a second one with a clone containing the empty plasmid. Sometimes the latter is generating some non specific bands which may have the same size as the specific expected one from the true clone.
The band intensity would depend on the amount of initial DNA. Since in colony PCR we manually pick up the colonies so it may not essentially be the same amount of starting material. That's why you may get different band intensities. Since your negative control does not show any band just the primer dimers this means that the quantity of DNA may be having some role in this.
I have had the same situation. My idea is that the petri plate from where you pick up colonies is already contaminated with (very little but amplifiable with PCR) the DNA fragment that you used to insert, say to plasmid. This is because the bacterial culture you used to transform with the DNA fragment of interest is now a mixture of DNA fragment+ transformed/ non-transformed bacteria, after transformation. This is what you spread on the plate. Therefore when you pick up colonies you are picking up some free DNA fragments with Agar as well. This may lead for different intensities with colony PCR bands. Also if you are doing the screening without using IPTG on X-gal/ antibiotic medium (E.g pGEM vectors), you may get false amplifications as well (positive colonies but no insertions).
Kosala Waduthanthri . I also have the same situation. When my petri plate have the mixture of DNA fragment+ transformed/ non-transformed bacteria, after transformation. How to get positive colonies with inserts. If so, can you elaborate the solution for this problem.
Thank you
Hi every one,
This time also i got same result for another transform analysis. Colony PCR was conducted with a negative (No plasmid) and a positive (Plasmid but no insert) along with clones suspected to be contain recombinant plasmid.
Negative - No bands
positive - 100 bp fragment as expected for vector primers
Clones - very pale band with expected size
I am very confuse with these results. I was tried with rapid screening to determined recombinants with size different. But highest MW band was similar to all including non recombinat. But banding pattern was different from cloned ones and the non recombinant. Here I have attached the gel picture of rapid screening. last two wells containing non recombinant plasmid.
Please help me to identify recombinants.
Thank you
I doubt about the plasmid extraction procedure you used. Did you use Alkaline lysis to extract plasmids from your clones? If you go through the colony PCR step first, you can figure out whether your extraction worked or not? Did you do colony PCR to your clones?
Hi Kosala,
yes I used alkaline lysis to extract plasmids. Control(Vector without insert-colony) was also used in same way. If the problem regarding with the plasmid preparation control also should affect, isn't it?
As I mentioned above I have done a colony PCR and got a very less intense band as expected size fragment. But my experience is it should give a very intense band.
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