PCR optimization failure - (Mar/26/2007 )
I've posted a topic under the 'product and vendor forum' regarding about optimizing a particular gene. I've tried several times but it failed so I wonder if anyone could tell me how could I start such as the concentration of reagents to use. Followed by if no amplification occur then how shall I proceed (e.g. change the concentration of 1 reagent at a time or several reagents?)? Is it alright if I were to change the conditions of several components at a time?
I've come across gradient programming to determine the optimum annealing temperature but can someone tell me how it works in the determination?
Another problem is, although there is no amplication for all tries but there are something like bands appearing for all including negative control. The bands are not sharp, but thick and faint. Can I know what is it? And lastly, how do I check if my template DNA is good?
I have only 1 month to accomplish so I really hope to accomplish this task. Hope as many people as possible could help me. Thanks.
I've come across gradient programming to determine the optimum annealing temperature but can someone tell me how it works in the determination?
Another problem is, although there is no amplication for all tries but there are something like bands appearing for all including negative control. The bands are not sharp, but thick and faint. Can I know what is it? And lastly, how do I check if my template DNA is good?
I have only 1 month to accomplish so I really hope to accomplish this task. Hope as many people as possible could help me. Thanks.
hello if you ar egetting bands even in the negative controls that means your PCR stocks are contaminated, the best solution is that replace your stocks, use new stocks.
This website is very useful for PCR problems. The author deserves a medal for taking the time to investigate the effect of every variable on PCR 
http://www.med.yale.edu/genetics/ward/tavi/PCR.html
Sorry for the late reply. My apology
Here it goes.
For MgCl2, try from 0.5mM to 5mM. But I personally think around 1-2mM is sufficient enough.
For primer, the recommended concentration is 0.6uM however, play around from 0.5-1.0uM.
As for DNA template, try this range, 10pg - 1ug. But for my case, around 150ng is good enough.
Be careful with your unspecific binding.
Hope this helps.
Cheers
Here it goes.
For MgCl2, try from 0.5mM to 5mM. But I personally think around 1-2mM is sufficient enough.
For primer, the recommended concentration is 0.6uM however, play around from 0.5-1.0uM.
As for DNA template, try this range, 10pg - 1ug. But for my case, around 150ng is good enough.
Be careful with your unspecific binding.
Hope this helps.
Cheers
Thanks! I'll try it and hope there'll be results. I wonder if it's my skills are lousy that's why unable to get the results the previous time but my supervisor has ordered a new set of primers and hope together with your suggestion it'll really works otherwise I'll be looked down when compared with my partner.
Hi guys,
I've tried all ways but still cannot! Is there a possibility that the microbe im using doesn't have the gene I'm trying to optimise? Or is there a problem with my skills? Maybe someone can point out the things not suppose to do but during my experiment process I did it but didn't know. I really really desperate for an answer.
have you blast your gene of interest with your bacteria's genome?
Another thing you can check is the DNA template. Not saying anything, perhaps maybe it is a wrong template to start with? 
I got involve in this project half way through so from what I know is the student involve in it didn't blast gene of interest with bacteria's genome. Okay, tell you what. The strain which I'm using now is LB400 and optimizing oxygenase genes. So there were no results even with the use of new primers, he said there might be a problem with the DNA template. In the sense that there are no oxygenase genes. So will it be better to perform DNA extraction again or get a new strain which contains oxygenase genes?
We can only convey rumors and generalities unless we know exactly what you are doing. Tell us in detail how the template was prepared, how much you are using, what gene you are looking for, what the primers are, what enzymes, buffers, water, cycler, cycle conditions, number of cycles, how you are testing the results, what you plan on doing with the DNA next. It amazes me that people think we can read minds and debug hard problems with absolutely no information about what is being tried or the results of those trials.
The template was inoculated a colony then performed DNA extraction using MoBio kit. Followed by carrying out PCR for amplification of oxygenase genes. The protocol is stated below:
10x buffer - 2.5ul
50mM MgCl2 - 0.75ul
100mM dNTPs - 0.05ul
1000ppm BPH1-F - 2.5ul
100ppm BPH1-R - 2.5ul
5U Taq - 1ul
T-DNA - 1.5ul
water - 14.2ul
PCR conditions
95 DC --- 10min
95 DC --- 1 MIN
57 DC --- 2 MIN
72 DC --- 2MIN
72 DC --- 10MIN
*30 cycles
The primers are supposed to amplify the oxygenase genes of the strain. After optimizing the PCR conditions for oxygenase genes, we'll proceed to environmental samples and determine the remediation capacity of these samples.