Hello Aleksandra,

Have you a positive control for your PCR amplification. It would help to understand what's going on.

We don't see any band for primer DNAs. It's not normal at all. Even if your PCR does not work, you should see the primers at the bottom of your gel.

Besides, I see some fluorescence (BET fixation) at the well of your gels, could your DNA be precipitated ?

Try to do a positive control.

Best regards

Pr Philippe Urban

Since the ladder looks good on your gel, there seems to be less chance of  problem with the gel electrophoresis. It appears that there is a problem with your PCR. You might be using too much of DNA in your PCR, which is visible on the gel wells. Quantitate your DNA extracts A260/A280 and assess the quantity and quality. Also try to check the PCR reagents using any  standard primer for host DNA amplification, such as b-actin/ GAPDH/ IL-IB etc for human DNA. If your reagents are Ok, then optimize your HPV viral primers.

best wishes,

SD

Hello, It seems that your PCR reaction is not working. There is no primer dimer at all. Try to do a PCR with a positive control. Also, check whether the primers are degraded. Additionally, your DNA may have some PCR inhibitors in it. So, check its quality and quantity.

I've run electrophoresis with positive controls and patients, please for comments. Products are not digested with restriction enzymes (Rsa I, Pst I and Hae III). It's screening . I used only the primers for viral DNA (MY09/MY11). I didn’t use the primers for human DNA (KM26/RS42).

Why I can't have this results with digested samples, where is my problem with patient, why I don't have results???

Here I have:

1 - 50 bp ladder

2 - NC

3 - PC

4 - PC

5 - PC

6 - PC

7 - PC

8 - PC

9 - positive patient

10 - negative patient

Hi

It is may be because your PCR dose not work and the fluorescence you see in wells belongs to unamplified genomic DNA and extraction yields which can not migrate further due to their massive size.

You mentioned that your positive control always works but i cant see any band on your gel. Is there a PC on this gel?

This can be due to several problems. May be your primers are broken or expired or your reaction materials dose not work. To check this you can perform a PCR using another sample and different primers.

Also check for probable high protein contamination in your extraction yield. 

Dear Aleksandra,

As I have mentioned earlier, your electrophoresis is ok. Problem is with PCR. I notice some points in your recent experiment. 

The MY09/MY11 primers are expected to yield an amplicon of around 450 base pairs of the HPV L1 region. However, compared to the 50 bp ladder in your gel, only the positive patient seems to yield the correct amplicon. Rest of the samples are giving an amplicon of around 150 base pairs. Hence the PCR needs to be optimized in terms of annealing temperature and/or primer concentration. for optimization you may use the positive patient sample as template. 

You are using the MY09 MY11 primer pairs for L1 gene amplification of HPV genome. this is degenerate primer system and widely used for detection of HPV genotypes, including the HPV16, 18 most frequently associated with genital samples. Although it is not clear what kind of samples you are examining, one possibility is that this primer system may not be sensitive enough for detection of HPV genotypes, which might be present in your patient samples. In that case you may try the improved primer system PGMY and GP5+/GP6+ primers for wider detection of HPV genotypes.

best of luck,

SD

Please go through the following references:

http://jcm.asm.org/content/40/3/902

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC229739/

http://www.mjafi.net/article/S0377-1237(15)00097-0/abstract

Aleksandra,

One more thing. You must do control PCR (using human primers) with all your samples for assessing the quality quantity of DNA and to rule out the presence of inhibitors in your samples.

For my amplification I used reagents from KAPA Biosystems.

Primers and restriction enzymes are from JENA bioscience. I dissolve the liophilised primers and diluted the stock solution of the primers (MY09/MY11) 1:10

My temperature program during amplification was:

• Initial denaturation (94°C for 2 minutes)

35 cycles of:

- Denaturation (94°C for 30 seconds)

- Annealing (55 °C for 1 minute)

- Extension (72 °C for 1:30 minute)

• Terminal extension (72 °C for 3 minutes)

Dear Aleksandra,

As all above mentioned, that had happened with my samples. I found DNA samples were totally degraded for unknown reasons. That might be a possible reason. I would also recommend as SD referred above, DNA concentration. Also, you should consider it the primers valid or not. Sometimes that could be a reason.

Good Luck

Marwan 

I've made same changes in the amplification. 

I made amplification with different annealing temperature (55; 55,5; 56; 56,5 and 57). 

Here are some photos of the gels. I think that 55.5 and 56 gave the best results. What do you think which one is the best annealing temperature... Also I made digestion for 1 hour on 37 degrees Celsius (and I stopped the reaction on 80 degrees Celsius for 20 minutes) with the amplificates on 55,5 and 56 digress with restriction enyzme Hae III and I got nothing  (I didn't use ladder).

For annealing temp. I cannot advise as I do not know which is 55.5 or 56C. I would suggest the second lane from the right side. Regarding digestion, I would suggest leave it for 2hr. If you get the same result that means your DNA is degraded.

Goodluck

Marwan

Dear Aleksandra Talevska,

I could not see just one photo right now.

Could you re-upload them again?

Regards

Marwan

Dear Aleksandra Talevsk, 

May be the problems in annealing temperature as you said. So, calculate the Tm for the primers pair you have used and try with that or best way to use gradient pcr. Hope you will get perfect result.

If DNA is not present then there is no chance of getting digested bands. In your supplied gel photo i am not seeing any positive band except the Ladder. !!

A little bit smear is seen (from down to up), it may be for the primers.

I think you need to look at this by starting with the samples and the DNA extraction. you will need to answer the following and follow the recommendations in the stated order to be able to figure-out the cause of what you are observing.

1. Are you using a crude DNA extract or a purified DNA extract as your template? usually crude extracts come with some impurities that may cause non-amplification. If so, you need to dilute the sample and test different volumes of your DNA extract during the PCR.

2. Were the DNA extracts stored well (freezer not fridge) and for along time? poor and long storage will lead to the degradation of the DNA in the extract. so try using fresh extract for the PCR if your extract have been stored for too long and particularly in the fridge.

3. Have you quantified the DNA in your extract? you need to at least have an estimate of the DNA concentration in your extract to sure that you are adding the right amount in your reaction mix based on the stated range for the primers you are using. not that too small and too large an amount of DNA will lead to non-amplification.

if these do help identify the cause, you may then check the annealing temperature by calculations using the Tm of the primers.

In my experience with crude DNA extract from paraffin-embedded tissues for HPV PCR I had to dilute the extract in order to achieve amplification. Two sets of dilutions were used overall for for most samples.