• are your primers sequence correct?
• are you using an annealing temperature in increment ramping?

It does seem like Andre is correct. The problem is in the ocr stage so please post your pcr cycle and number of cycles. Do you have any other primers to prove that the template dna is amplifiable? How old are the primers and what are they stored in?

How much primer are you using and how much magnesium.

Do you have an od260/280 ratio to assess the quality of the template dna

Andre, the primer sequences are correct as far as I can tell. I have checked and double-checked.

Paul, I have run the template DNA with the invA and invE primers previously to confirm as Salmonella, and the template DNA amplified fine then.

The primers are only a few months old and stored in a -20 degree Celsius fridge and resuspended in autoclaved DI.

I am using 1.5 ul of each primer (20mM). I am using 1ul magnesium (25mM).

I have used template dna with the following 260/280's: 1.53, 1.78, 1.54, and 2.13.

I have used four different PCR protocols from three different papers/sources:

1.) 94°C for 2min; 30 cycles of: 94°C for 1min, 45°C for 1 min, 65°C for 5 min; and a final extension of 65°C for 15 min

2.) 95°C for 5min; 30 cycles of: 90°C for 30sec, 45°C for 1min, 72°C for 1min; and a final extension of 72°C for 8min (where I got some bands)

3.)95°C for 5min; 30 cycles of 94°C for 3sec, 92°C for 30sec, 50°C for 1min, 68°C for 8min; and a final extension of 68°C for 15min (original protocol I was given)

4.)94°C for 5min; 35 cycles of 94°C for 1min, 52°C for 1min, 72°C for 2min; and a final extension of 72°C for 5 min

All extensions were only for one cycle.

Thanks for the replies!

Ralph,

Why did you do just one cycle? You won't be able to detect amplification with just one round.

The extension temperature will depend on the enzyme you are using. Some Taqs work at 72oC and others at 68oC (you should check the one you have)

I believe the problem with your experiment are:

• low cycle numbers (you should go for 35 cycles)
• low extension time (1 min is not enough)
• annealing temperature

I found this article that uses the same primers you are using for Salmonella. Take a look.

"The PCR was performed in a 50 µL solution containing 1 µM of each primer, 5 µL of 10X PCR buffer, 250 µM dNTPs, 3 mM MgCl2, and 3.0 U of Taq DNA polymerase (Sigma Aldrich). The PCR conditions were one cycle at 95°C for 10 min, followed by 4 cycles of 5 min at 94°C, 5 min at 40°C and 5 min at 72°C and then followed by 30 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C, and the last extension at 72°C for 10 min."

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081315

thank you Ralph there is much work there!

I am still unsure on 2 aspects...the magnesium concentration is critical and you do not give the final pcr volume but I think that the final magnesium should be 1.5-2mM. I would not store primers in water as it absorbs co2 from the air and the acid depurinates the single stranded primers. TE is better. Also your cycles seem very long....how long is the amplified product...1 minute is long enough for 1000 bases and annealing times seem rather long...a few seconds is good but I do agree with Andre that more cycles are needed...35 or 40 to start with then change conditions once the pcr is working Try starting at a magnesium of 2.5mM which should work dirty but at least you have a product to clean up

Andre, when I said that I used one cycle, that was in reference to the final extension in each protocol. However, there were 30 cycles of every extension. I think my write up was a little misleading.

I will double check the Taq. Thanks!

Also thank you for the paper. I will definitely give that a try. It is a lot different protocol than I have.

Paul, my final PCR volume has been both 25ul and 50ul (double MgCl2). The amplified products are between 400 and 500bp if I remember correctly, but I am having trouble finding where I got that number from. I know there is a way to BLAST primers and find the target size, but I have forgotten how. I will post if I figure it out.

Thanks!

Check your magnesium I think  you are not using enough. Start with 25ul RXN volume and 2.5mM Mg and lowere the annealing temp .Then when you get dirty /many bands clean up with temperature or DMSO.>If all else fails make your primers longer…it makes annealing temperature higher and the template has less time to re-anneal so pcr works better

Thanks Paul! I will definitely try what you are saying. I will let you know how it turns out.

Paul, do you have any recommended conditions for running the electrophoresis?

 Ralph, that will depend on your gel aparatus. Mesure the distance between eletrodes and use something between 4-10V/cm. I prefer running in TBE and low voltage (gel looks better).

it depends on how good you want the results. The slower you run the samples the less the bands broaden because there is little heating of the gel so less sample diffusion . I would run in TBE at 5volts/cm of gel . If you use orange G dye it runs faster than all pcr product but the common blue dyes run  at 200 bases or less so run the blue dye about halfway down the gel ( about 2-4 cm) and check the product

Andre and Paul, thank you both for your response! I will incorporate what both of you have suggested and let you know about the outcome.

Alright, so I followed a combination of both of your recommendations. I got the MgCl2 up to 2.5mM, lowered the annealing temp to 40°C, increased the cycle times to 40, and ran the following PCR protocol:

95°C for 10 min, 40 cycles (94°C for 30 sec, 40°C for 1min, and 72°C for 2min), and a final extension time of 72°C for 10 min

I ran the gel at 5v for 6 hours, and I got nothing at all except the ladder.

However, I ran another gel at 50v for 1 hour, and I got some amplification to appear. It is not pretty at all and looks nothing like other ERIC-PCR results. A lot of bands are faints, but it is the best run that I have had so far. I have attached it with the other papers and my protocol that I was given for you guys to look at.

Maybe you can help determine the issue if you have more info.

50V for 1h run is a good way to go for that gel size, you should stick to it.

Have you tried increasing extension time? I can see small bands but not larger bands. I've seen some protocols that use up to 5min extension time.

Are you using EtBr in your gel? If you are that would explain why the lower part of your gel is not as bright as the top part. EtBr runs upward (in the opposite direction). Can't you make an EtBr bath to soak your gel? It will look better.

I have tried those longer extension times up to 8 minutes even.

I do use EtBr in the gel. I can make the EtBr bath afterwards, but I have never done it that way. What percent EtBr? What liquid for the bath? How long should I soak?

I have also ran larger gels at lower voltages, so I am not restricted by gel size. It is easier for me to make a small gel to test for any amplification at all. Is there a size that you recommend?

Also what do you typically set mAmps to?

You may be having difficulties seeing the more faint bands. How is your gDNA? Is it good quality?

You can make a solution of 0.5ug/mL EtBr in ddH2O and soak your gel for 15 min. New solutions are strong so if you get a high background you can soak it in ddH2O for a few minutes. You can reuse the solution for a few days (just keep it protected from the light). The older the solution the longer it will take to stain.

I always run gels at a fixed voltage. Small gels I use 50V and larger gels I use 100V.

gDNA information is as follows: 1.78 260/280 and 24 ng/uL by NanoDrop. I also ran it on a gel and got a bright band at the top of the gel. There was no smearing indicative of shearing.

My questions about voltages and gels comes from the literature being mixed on those issues. I have seen 40v for 12 hours, 50v for 6 hours, 120mV for 1 hour, 5v for 4 hours, etc. I am aware that different machines and different gels. However, they do not always list gel size, gel dock brand, pcr machine brand, etc.

2 things:

first I am concerned about primer ericr..in Lankau paper there is no C at the 3' end. If the 3'end base is wrong there will be no amplification. Please check this sequence.

secondly Magnesium still worries me. If you thaw magnesium solutions incompletely or do not thoroughly mix it when thawed then you get strong Mg released in the early stage of thawing and if you pipette some of this out then the remainder is weaker than labelled. If your primer is correct and you are getting weak bands now then borrow someone elses MG and use 2.5ul of 25mM in a 25ul reaction or use twice as much as your last experiment of your MgCl2 solution and see if things improve.

The voltage will varie acording with the distance between electrodes. Mesure de distance between electrodes (not gel size) and always use 4-10V/cm. This will avoid heating. You can follow the duration acording with the bromphenol line in your loading dye.

@Paul

I checked the primer in other articles and some finnishes with A and others finnishes with C. In normal PCR it's better to end a primer with C or G. Do you think that finnishing with A could help amplify the other bands?

Paul, I am working directly with Lankau from the Lankau paper, and she originally sent the SOP which is the word document that I attached second on that post. However, I have seen what Andre sees as well, some ending with A and some C.

I will try a new batch of magnesium with better mixing and thawing  techniques.

My question for you, Paul is the following: does the gel run above show amplification that rules out a primer problem even though it is not very good?

Andre, I will also try a new gel without EtBr.

That last run is one of the more promising.

Thanks for the help again!

thanks Andre...the C is only a problem if it mismatches with the target genome....if it matches the target genome then the extra base is a very good thing because these primers anneal at a low temperature which is bad for pcr. The higher the annealing temperature the less chance the denatured dna has time to anneal back together which would spoil the efficiency of the pcr. My ideal pcr goes 94,72 two steps but this does need long primers.

Ralph...there is no sign of primer dimer and the bands look clean and well separated so the primers are working well but I think you have not loaded enough sample on the gel. This would brighten the bands. If you run a gel without etbr it will run 10% faster. You may want to investigate size standards with smaller fragments  to give you a better idea of the accurate size of your amplimers at some stage. A 10bp or 50 bp ladder perhaps but your results are looking very much better

also while getting the pcr conditions right dont run the gel so far...the bands will look brighter and I think that what Andre meant was that you could run the gel normally but improve the band intensity by soaking in  etbr when most of the etbr has moved up the gel during electrophoresis. Also do not illuminate for too long under UV light before taking the picture...it is possible to photo bleach the bands with too much UV and they can become invisible. regards

paul

Paul and Andre,

I ran a gel without EtBr in it and soaked it in an EtBr bath afterwards. I ran it for 35 minutes at 50V (the distance between electrodes is 13 cm).

As you can see, it has smearing.

From left to right, the gel is as follows: 100kb ladder, 1000kb ladder, E. coli, Environmental Sample with Salmonella, Stock Salmonell in 5 and 6.

It is still not working like it should, but the bands are brighter.

Ralph

Ralph

Maybe you already had the smearing before but since your gel was more faint you couldn't see it.

Have you tried an annealing gradient? You can take one of your samples and run the same reaction but at different annealing temperatures.

How are your primers? Are they old? Have you made dilutions from your stock and kept them to avoid freeze thaw cycles?

How much DNA are you using?

What were the annealing and extension time for this particular experiment?

There is either a loading or a visualisation problem here. The samples compare well with the size standard but the standards are very weak. Are you loading too little or is there a problem with the filter or UV lamps or exposure time on the transilluminator. Give the gel a longer exposure time on the camera but not too long on the UV light box setting up the picturebecause that will weaken the signal I would soak that gel again in stronger ETBR to see if it makes a difference. Also I would load more of the pcr product. Smearing is not ideal and maybe suggests that one primer is not annealing quite as well as the other but the bands are readable and can be sized so you are really getting there. Now that you have stronger bands you can play with parameters to improve the pcr such as raising the annealing temperature, primer concentrations and Mg again because you know that all of the other parameters are working. You could try using more primer just in case one is degrading as an early experiment

Andre, I have not tried an annealing gradient.

Primers are two months old. I have made dilutions from my stock of primers. Stock was reconstituted at 100mM. Stock is at 20mM.

I have used DNA in amounts from 10ng/uL to 100ng/uL to 1000ng/uL.

From the original protocol, the annealing temp is 40 C for 1 min, and the extension was 72 C for 2 min.

Paul, my main concern is that the banding that is showing up is not consistent with the type of banding that one would expect to get with ERIC-PCR.

For instance, this is representative of the banding patterns that I should see.

ok I think that yours look better....all of the smaller bands are constant in the representative picture and your bands look like the variable bands at the top . Clearly there is a huge size difference between yours and theirs so I am wondering if they mislabelled their ladder which actually has 100bp bands up to 1kb and not lots of bands of 1000bases difference in which case yours and theirs look more similar. Have you tried blasting your primers to see if they are a few hundred or a few thousand bases apart but at least I now understand why the extension times were so long. It is possible that some of their extra bands are non specific bands from human DNA so you will not have them

Ralph

Have you seen these articles?

http://onlinelibrary.wiley.com/doi/10.1046/j.1472-765X.1997.00162.x/pdf

https://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/18/25.pdf

I think you should increase the annealing temp (try a gradient) specially because primer ERIC2 has a very stable hairpin around 34.5oC which can keep you from amplifying. If you consider the articles, increase the elongation time (like the original protocol you were given) may also help.

You said some days ago that the original protocol was:

95°C for 5min; 30 cycles of 94°C for 3sec, 92°C for 30sec, 50°C for 1min, 68°C for 8min; and a final extension of 68°C for 15min

is that 94oC for only 3sec correct?

Hi Ralph,

Here is my two cents worth:

I would use TBE buffer for electrophoresis rather than TAE. It gives much better runs. I would also run 2% agarose at 100 volts/50 ma till the bromophenol blue marker runs about 10 cm.

Always stain afterwards, not in the gel, and I would also swap to GelRed or some other non-ethidium stain.

Inclusion of a PCR stabilizer like GeneReleaser always improves yield and reproducibility (see attached paper with protocol), particularly of the higher molecular weight bands.

If you are just interested in identifying clonal lines for epidemiology/tracking, then you might do better with another primer. ANY primer of 20-24 nucleotides will generate complex PCR banding patterns when you use the ERIC PCR program. That's because it is not a specific PCR, but a very reproducible version of the RAPD procedure (See attached paper)

Hope this helps,

michael

Ralph, besides the mentioned concerns, I have some additional comments and suggestion. First,Nanodrop is not a very precise way to measure the DNA concentration although it's very convenient. Did you run a gel analysis of your gDNA? How bright and how integrated it is? ERIC-PCR and BOX-PCR are typically not very demanding of DNA quality as far as I know. I suggest you stick to the template amount when you successfully amplified other gene markers from your strain. The best template amount for me is 10ng for a 50ul volume. And do NOT use DNA template up to 1ug. It will only inhibit the amplification.The Mg solutions need to be THOROUGHLY dissolved before using (most  Mg2+ could be locked in the ice). I suggest you use a final concentration up to 4mM of Mg for ERIC-PCR.

 You can also try two-step PCR ：95°C 3min of predenaturation, then （95°C 30s, 68°C 1min-2min) X 30; 4°C archive 

Good luck.

Andre, I think you might have been right about a longer extension time. I increased up to six minutes and got lots of banding. The bands are not very clear, but I am in the process of longer timed run (4 hours) on a longer gel in hopes to get better banding.

Michael, I have been running the blue marker that long. The problem is that most protocols run these gels for multiple hours. Also, this is the second set of primers I have used. I originally started with REP primers and not ERIC. I need to make one set work.

Fang, I am aware of Nanodrops shortcomings. However, it is what I have available. I ahve run the gDNA. I got one bright band and two faint ones.

Paul, I thoroughly thawed the MgCl2 this time. Maybe that was a key issue.

Any thoughts on how to clean all of this up a bit?

I also need to play around with the camera. I am not very good with using it.

By the way, thanks for all the help that everyone has provided. You have all been pivotal in the breakthroughs that I have had thus far.

Hi Ralph

Several things can cause that smear. Some of them I already asked you (primer quality, excess template, etc). Now that your experiment is on the right way you can try to solve it. 

How much enzyme are you using? Low annealing temperature is also a factor. Have you run that gradient I mentioned?

I'm glad to help. I am also learning. Just send me some Atlanta style fried chicken and cornbread and we are even! LOL

Ha, ha, ha! For the all help you provided, I would send you all the fried chicken that you can stomach.

I just ran a four hour gel.

I tried to label it better than the others. Nothing showed up particularly bright. If I could somehow manage to make them show up better on such a long running time, I think that I would have exactly what I am looking for when it comes to ERIC.

Ralph

How much (volume) are you applying in your gel?

What is your gel percentage? Are you using TBE? Why are you running the gel for so long? A gel with that size you can run at 130V for 1.5h

Andre,

I am applying 20 uL total (16 PCR product and 4 dye).

My gels are 1.5%. I am using TAE. I need to make more TBE.

I ran that long because the protocol that I was given, and the papers that I am working from all have long running times (varying anywhere from 4 to 12 hours).

The first gel from the batch was run for a shorter time.

I guess that I figured all the variation in running times from various sources had something to do with getting better runs.

Ralph

For a 1.5% gel bromophenol should be around 400bp. Try running your large gel for 130V (fixed voltage) until bromophenol reaches 2/3 of the length. Perhaps the papers used longer times but small voltage and also a different electrophoresis system. If you check your gel under UV light and it hasn't been long enough then you can CAREFULLY put it back at the tray an continue the run.