I have done PCR many of the times with the same DNA, sometimes I got good results, sometimes they did not show any specification.
What is the problem for this condition or do I need to change any reaction?
When you say "Did not show any specification", do you mean nothing appeared in the lane when you run a gel; or you get a smear, or...?
Hi, I have some questions before putting my suggestions, for how long you have been doing PCR? If u r new then probable reason is error in handling (pipetting). Same DNA means same sample? Got good result, u mean band in agarose gel? what is the product size. I guess u were getting the target product in some reactions but not while repeating the same.
Try these steps
1. reduce the chance of any contamination (template) in primers or water or any other materials.
2. what is the annealing temperature and what is the difference between ur Tm and Ta? did you do gradient PCR? better select the highest possible annealing temperature.
3. Are you sure the good result you said is really good? did you run a positive and negative controls in parallel?
4. what is the template concentration? too high is not good.
5. what is the product size, set denaturation and annealing duration shorter and time for extension according to the product size.
6. What is the gel concentration? if product size is smaller, use 2-2.5% agarose gel.
hope these points will help u in getting better result.
Satheesha already give some hints what reason for a negative PCR can be. An other reason can be that you maybe use different cyclers with the same program. The problem by using different cycler is that the heating and cooling times are different. If you have for example a PCR which work good at an annealing temperature 60°C the real annealing is 58°C. If the cycler need for example 30 sek to go from 57 to 59°C the primer have about 10 sek for annealing. So maybe the time is not enough for annealing = no result. If you use then an older cycler which need more time to go from 57 - 59°C the anealling temperature at 58°C is longer = reasult.
So it is very important to use allways the same PCR cycler for difficult PCRs.
Always include negative (no template) and positive (template that you know works) controls in your PCR reactions. Thereby you can immediately rule out false positives and show that your reaction mix works. If you want to do the same PCR on many samples it is a good idea to make a master mix (PCR mix (minus template) x number of samples x 1.1). Thereby you ensure all the samples get the same mixture of PCR components.
I have done for aforementioned gradient tm, positive and negative control, and chosen 50-60C temp, diff cycles,etc., except 2.0-2.5% of gel concentration. moreover i have worked that DNA ~2.7kb, ant it has cloned and sequenced. here I want to amplify again that same condition with different isolates (Virus) and also same taq polymerase but it is not showing any result. some times it has been come and some times not. My question is Do I change any temperature or change master mix reaction.
Check whether primers are degrading... different isolates means did they are field isolates??? viruses acquire mutations rapidly..it may be one of the reason that, because of the mutations at primer binding region may decreases the affinity of primer towards template..
Deivamani,
Please write your primer sequences, PCR conditions and components that you used and product size. These information will help me to suggest you.
Few questions:
What is your host plant?
Virus?
Did you use any internal control to make sure that you have successfully extracted the RNA?
One step or two step?
cDNA synthesizing?
Dear Deivamani,
Check your primers for repeating your samples, which get the best results as you want, its confirm your primer degrade or not. Different isolates especially field, if you are using, its not necessary to got the same products from different one. you may check the ready made Master mix, if you are new its possible pipetting errors or your isolated DNA are contaminated.
Make sure it's not a problem with the template. If the problem is a few mutations in the primer binding site(s) I think a lower annealing temperature will help (but you will probably get some non-specific amplification as well).
thank you for suggestions.
Crop. tomato
Virus: ToLCV
Pcr condtion: 94 for 5m, 94 for 30s, 57,1 for 30s, 72 for 2m, 72 for 10m
primer sequence BAMHI restriction sites containing forward and reverse sequence.
product size 2.7kb
It could be that your elongation step is too short. The general rule for Taq polymerase is 1 min/kb, so perhaps you can try:
72 C for 2' 45" (or 3' )
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