I am trying to do some basic mouse genotyping, but am having problem with a thin band being seen in the water. I have 4 other genes that I routinely genotype and never have had this issue. I use the same water, taq, tubes and pipettes, but this is the only gene I have had trouble with. I was concerned that it may have been the primers themselves so I ordered another pair, taking care not to use the same water or pipettes to dilute them. They are currently at 100 micro-mole concentration and I use .1 microliters for every sample. After ordering different primers I am still having the issue. I have also tried using another bottle of taq, different water, different pipettes as well as a higher fidelity taq. The higher fidelity taq seemed to make the band skinnier, but it is still in the water (neg control), but it is not ideal for me to use a more expensive taq just for genotyping anyway. This pcr should result in a 550 bp band for the mutant and no band at all for the wild type. In the attached picture, the last band is the negative control.
PCR conditions have been :
94'c 3mins
94'c 30sec
57'c 30sec
72'c 45sec
37cycles
72'c 5mins
The Tm of the primers are 64.9 and 63.5
In this case what i would suggest you is that :
1) just load say 5 micro-liter of each of your component like dNTPs, water, forward and reverse primers, your buffer, MgCl2(as you have changed Taq. so theoretically it is not contaminated) , and go for Agarose gel electrophoresis...and the contaminated sample should give the same band.
2) and second thing is that you just repeat one the earlier reactions(those reactions which are earlier not giving bands in negative control) with the same components you are using for the last primer set reaction. and check if they are giving the contaminated band.
hope this will give you your desired results..
all the best
decrease the number of repeat cycles to 29X. The contaminating product is also of a different size and the intensity too is way weaker than the correct band. I would not be too worried, however I would UV irradiate all tips, pipettes, water. I do not know if it is feasible for you to switch to a different Taq but I would recommend DreamTaq Green PCR Master Mix (2X) # K1081. Very cheap and very efficient, and time saving. You just use 7.5ul/reaction of 15ul (1ul primer mix, 1ul template, 5.5ul water and 7.5 master mix, and best part you can directly load into gel. Goodluck
I think you have two problems (since you have two non-specific bands)
1) Your primers are amplifying something random in the genome when your gene is not present (= larger molecular weight band)
2) Your primers are either dimerizing or picking up a random spec of DNA to amplify (=thin ~500 bp band).
Both problems are likely caused by your conditions being way way way way too permissive.
You don't need to buy a bunch of new reagents.
Try the following first:
1) Decrease your cycle number to 30
2) Increase your annealing temperature to 62
3) Use 20X less primer. (For a 25 ul reaction I usually use 0.5 ul of a 12.5 uM stock)
Also, despite the non-specific bands, your genotyping result is very clear. (samples containing the mutant allele are 1,5,7,8,9,10,15,17,18,19,20,21,22)
Thank you Prakash, Bharat and Shanni! I will try a couple different things starting with decreasing the amount of primer and the amount of cycles and let you know how it works out. I really appreciate everyone's time!
So I decreased the cycle number to 29, changed the annealing temperature to 62 and used about 1/2 the amount of primer and there are no more mysterious bands! Thanks everyone for the suggestions!
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