Problem for PCR - (Jan/22/2014 )

Hi,

 

I did couple of runs of PCR to detect Chrysanthemum stunt viroid (CSVd) in chrysanthemum plants. Expected size: 313

 

Included;

12.5 ul master mix (Fermentas)

1 ul forward primer ( 10pmol)

1 ul reverse primer (10 pmol)

8.5 ul water

2 ul cDNA

 

Conditions

94:4 m

52:45 s

72: 45 s

35 cycle

 

I used 20 samples, results were attached (Figure 1). When you look over figure 1 cautiously, u can realize hardly visible bands line 2, 4, and 12.

(The picture is not very clear, maybe you can't see bands, but they are.

 

Then I did a PCR using 2, 4, 12 samples again increasing the cycle to 40. Results were attached (Figure 2). When you look over figure 2, you can see 2 bands sample 2 and 12.  (Line 1: 2, Line 2: 4, Line 3: 12)

 

So what went wrong in my PCR and what sould I do?

 

 

There's no figure attached.

In your PCR conditions the repeated denaturation step is missing.

I dont see the attached photo. But observing the conditions, if your denaturation step at 94C is 4 minutes every cycle, that is WAY too long. Are you doing a two-step cycle? Most modern enzymes only need 10-30 seconds for denturation after the initial step. If you are doing a two-step protocol, then ignore this, but it is something to look at otherwise.

 

You could also try raising the concentration of primers. Those seem a little low.

I can't upload the files. System gives error "you are not permitted attach this file"

 

Denaturation step is not missing, I have forgotten to write it. It ıs 94 C, 45 second.  4min at 94 C is for start to activate taq polymerase.

 

the problem is hardly visible bands when using 35 cycle in conditions given above. When I increased the cycle to 40, I saw two band, one of them is in true position (313 bp), the other one is between 100-200 bp.

Then it might be too large or an inappropriate file-format.

 

Anyway perhaps you can increase specificity e.g. by reducing the annealing time (e.g. to 30 s) and/or increasing annealing temperature (if possible)

I took primers and PCR conditions from a published paper, and just applied it without any changes.

 

melting temp. forward 58; reverse 62

 

In my opnion, anneling temp. that I applied is too low. It is better to increase as hobglobin said. And do you think increasing primer concentration is good?

 

Thank you

It's surely worth a try. Anyway depending on the aim of the PCR I'd not spend too much time in optimisation, i.e. if need the 313 band to detect the virus and get it with 40 cycles and can distinguish it easily from unspecific ones, it should be more or less okay.

If you need the product later for other applications then of course you should try to make the PCR more specific.

I will isolate it from the gel to clonnig and characterization. I am not sure that is it ok for this purpose? because I will use rounds of band, it can be ok, maybe not.

 

I'll make a last try with modifications including increasing anneling temperature to 55, decreasing anneling time to 30. 

I'd suggest that you try a range of annealing temperatures. I don't think annealing time will have much effect. At 40 cycles, you are likely amplifying junk, not your desired product. I don't trust things that amplify after about 34 cycles.

A major difficulty I often see is adding too much template. Try doing serial dilutions of your template: 1:10, 1:100

How confident are you that your cDNA preparation is working? Is there a control gene that you are testing?

Is this a RNA virus, or can you amplify directly from genomic DNA?

I thınk you're right, there was just a hardly visible band when used 35 cycle, then 2 band were seen and one ofe it is junk probably in 40 cycle.

 

I did not understand how can much template effect and cause this problem? Could you please explain?

 

this is a RNA virus so I can't apllify directly from total nucleic acid.