Hello, I am currently having issues optimizing my PCR reaction. I am unsure if I am getting amplification when using 250 ng of template due to my template only control having a similar band. My expected product size is 234 bp. I have tried optimizing annealing temperature, amount of template, MgCl2 concentration, and tried adding DMSO. I am looking for any ideas on what the issue might be. Here are my conditions: 0.2 mM dNTP, 1.5 mM MgCl2, 0.5 uM primers, 2.5 units of GoTaq polymerase, and 5% DMSO. I run the samples at 95C for 2 minutes, 95C for 30 seconds, 52C for 30 seconds for annealing, 72C for 30 seconds, and 72C for 5 minutes for 35 cycles.
I have attached three recent experiments. When looking at gradient optimization 2 there is clearly 2 bands, and suggests that we are getting some sort of amplification. However, we have not been able to replicate this. Thank you.
- It is obviosuly target gene and primer specific but
50ng template and 250ng template seem to bear no relationship. So are the amounts of template (only) loaded into all lanes the same?
What is the predicted Tm of your primers?
- It is obviosuly target gene and primer specific but
hi
1-I would like to select 20 ng of template .
2-check template quality by housekeeping genes primer (example GAPDH or Beta-actin ,...)
3- Try to use fresh template with the high purity and nice quality.
If the problem is not fixed, you need to design and use a new primer set ,because your band intensity is not good enough.
https://www.qiagen.com/be/resources/faq?id=1e64371a-1cec-4d0b-a049-ca570f909c3a&lang=en
good luck
Thanks for your comments!
Geoff Margison - The Tm of my primers are 57C and 59C. Also, the template only loading lanes contain the same amount of template used in the other lanes except with no primers. So, they only contained 0.1 ng, 1 ng, 5 ng, etc. The purpose of that experiment was to try to optimize how much template we needed to add, and to try to find out why we were getting a band at ~200 bp in our template which made it hard to determine whether we were getting amplification or if it was simply our template.
Laurence Stuart Dawkins-Hall - Thanks for taking the time to write out your thoughts! The Tm of my primers are 57C and 59C. I picked 52C for my other experiments because that was the only temperature that we saw a band we were looking for, and it fit with the idea of Tm - 5C. We tried a temperature gradient from 46 to 64 (forgot to label those values in the image above). We have not tested our template using other primers, but it seems like we will need to order some. I will reduce the concentration of primers and try another gradient.
Also, someone had left a comment (now deleted) about my template looking degraded. Should I make another sample? I also got a suggestion to try RNase A treatment, but it is my understanding that RNA would be degraded during the high temperature PCR cycles anyways.
- Yes making a fresh template would be a good idea
- How are you making it ?
So what exactly is the template?
If its genomic and you havent included an RNase step, your input isn't DNA. Indeed it might be mostly RNA!
Thanks for the quick responses! I did not perform an RNase A step, and I will try that today. The template I used in the above experiments was from an MCF7 cell lysate, and the DNA was extracted using a Phenol-chloroform extraction.
Mcf7 in my experience should give clean DNA
Having phenol chloroform extracted your cell lysates ppt with 1 vol of isopropanol and having then spun wash pellet x2 with 70% room temp ethanol. That will ensure desalted DNA
Hello, Dr. Adam
There may be many probable reasons...Try using these steps..I hope this will decrease smearing...
1) Decrease the enzyme and mg conc.
2)Increase the denaturation time and temp.
3) Reduce extension time
4) Decrease the cycle number
4)Decrease the possibility of DNA carry over
You should check:
a) promer specificy
b) PCR conditions (esp. annealing temp. should be high enough to warant specifity, but not too high to alow annealing itself)
c) no solvent, such as ethanol is in the sample
besinde some other points.
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