Did you include a positive control and show one band? Positive control will tell you that your PCR set up and conditions have no problems.

hello Yuan,

thank you very much for your response, and yes  I included a positive control but there is no band are showing for + control and samples, as well.

here details about what I did it:

(I had 6 samples of mites and + control is wolbachia cell line)

the ingredients for PCR contain of: the total of reaction is 25µl

Reaction buffer 2.5µl = reaction mix is 2.5 x6= 15µl

dNTPs 0.5x6= 3 µl

Forward primer 81(10µm)  (1x6= 6µl

Reverse primer 169( 10µm)  (1x6= 6µl

AmpliTaq (0.2x6= 1.2µl

water 19.8x6=118.8µl

DNA Template (1µl)

PCR Cycling was:

95c   3 mint

95c    30s

54c    45s

72c    1mint       2  34 cycles

72c  10 mint

Hi Samia,

1. If your positive control has no band, that probably meant that something is wrong with your PCR setting up, conditions, or your PCR machine. Positive control is supposedly to have a band.

2. What is the Tm (melting temperatures) of the two primers?

3. Do it again with carefully adding your 'master' mix to each tube. Consider to add 'PCR additives' such as DMSO in the PCR reactions. DMSO improves PCR efficiency and specificity. It is suggested to add in PCR reaction if your template is GC-rich.

Hello Yuan,

thank you ver much. 

yes. there is no band in positive control and really i don't know what is wrong?

about melting temperatures, I tried 54c and 52c but also no band.

finally, could you please explain more this point DMSO because i did not understand?

Consider to add 'PCR additives' such as DMSO in the PCR reactions. DMSO improves PCR efficiency and specificity. It is suggested to add in PCR reaction if your template is GC-rich.

thanks again.

samia

Wsp 81f&r primers? - ResearchGate. Available from: https://www.researchgate.net/post/wsp_81f_r_primers#578dada3cbd5c2845221a5f1 [accessed Jul 19, 2016].

1. 52C and 54C you set for PCR were 'Ta (annealing temperature)'. It is not 'Tm (melting temperature'. Each primer has its own Tm (this data is shown on the data sheet which comes with your ordered primers). Or, you can estimate Tm of each primer by using online program. Very simple.

2. Please read the attached article carefully about DMSO for PCR additive. The article explains and shows experimental results of adding DMSO in PCR reaction.

thank you very much and  the Tm (melting temperatures) of the two primers are for WSP 81F =57.6 AND WSP691R= 49.1.

Hi Samia,

The Tm of your WSP691R primer = 49.1 oC, which is lower than the Ta (52oC) you set. So, there is a possibility that this primer did not anneal to the template, and caused no PCR product amplified. You can try PCR again with Ta = 49 or 48oC.

Please do it again and let us know the result.

1. Also how long is your expect PCR product? I saw you set the 'extension time' = 1 minute.

2. By the way, you said that "I had 6 samples of mites and + control is wolbachia cell line". Do you need 7 PCR reactions (6 samples +1 control)? If so, why did you prepare 'master' mix only for 6 tubes (x6, as you stated above)? You also need a 'negative' control (use water to replace DNA). Negative control should yield no band.

Hi Samia,

Have you got a chance to re-run a PCR with a different Ta yet? If so, have you gotten anything.

You can try the Ta value I suggested above, or you can run a 'touchdown PCR' (If your PCR machine equips that program) to find out the best Ta (produce a good PCR band).

Hi Yuan,

Thank you very much. The size of PCR Product from 590-632bp. as well, could you please explain to me this as you said above( you can run a 'touchdown PCR' (If your PCR machine equips that program) to find out the best Ta (produce a good PCR band).

I appreciated.

Hi Samia,

In regular PCR, the annealing temperature (Ta) is the same during all PCR cycles, while In touch-down PCR (or TD-PCR), the initial annealing temperature (Ta) is higher than the optimal Tm of the primers, and then Ta is gradually reduced over subsequent cycles until the calculated Tm temperature or some degrees below is reached, much like the touchdown/landing of an airplane. Because there are several different Ta involved, hopefully, some of the Ta will give us a PCR band.

Touchdown PCR enhances the specificity of the initial primer–template duplex formation and hence the specificity of the final PCR product.

I attached an article explaining touchdown PCR (see attachment). It is from http://bitesizebio.com/2203/touchdown-pcr-a-primer-and-some-tips/

By the way, have you got a chance to re-run a PCR with Ta=49C yet? If so, have you gotten any band? Could you post your gel for PCR results?

This is one study example of using touchdown PCR:

Maybe his calculated optimum Tm = 62C, so he set his 'touchdown Ta' from 67C to 57C.

http://www.ijmm.org/article.asp?issn=0255-0857;year=2015;volume=33;issue=2;spage=271;epage=273;aulast=Hallur

hello Yuan,

thank you very much and I really appreciated. I am a  PhD student  in the first year so you are help me a lot. 

regards

samia

Hi Samia,

Looks like that these two pairs of primers WSP81F and WSP691R have been wildly used as a standard primers for PCR by quite a few researchers (see attached paper, yellow highlights). It should not be that difficult to amplify a PCR product by using them.

I think you should work on the 'positive' controls first and ASAP, if the positive controls don't even work, you will have problems with those 'sample' PCR. Remember to add a 'negative (replace DNA with water)' reaction.

hello Yuan,

I re-run a PCR with Ta=49C and 55c and I got a band in positive control. Can you see the attached file please .

many thanks

samia

Hi Samia,

Very good and clear band for the positive control and no band in the negative control. And, the size is correct (match your expected size, ~600 bp). This is a good result. This means that your PCR conditions, PCR machine are in good conditions to do your experiment. Congratulations!!

1. Look like both Ta temperature are amplified. In some cases, Ta with a few degrees over calculated optimum Tm can still amplify a PCR product. That is well document. So, probably last time (you use 54C), something was not right (most likely the reaction ingredients set up), and resulted in no bands.

2. Now, you can proceed to do your 'experimental' samples (set up: Samples + positive control + negative control). Please update us your results after you done.

By the way, one thing I would like to point out for your future references:

The question you wrote on RG was: "Wsp 81f&r primers?" The first impression people will get from that is that you are using WSP81f and WSP81r for your PCR, but actually you are using WSP81f and WSP691r for your PCR amplification. Each primer has its own name. The name WSP81r might actually exist on documents somewhere. If so, that will confuse the researchers/audience. To make audience more clear, it is better to use WSP81f/WSP691r in your presentation/papers in any forms.

Look like that this pair of primers (WSP81f/WSP691r) are one of standard primers in this field for specific PCR amplification. So, if you give a talk and use WSP81f/WSP691r, those people in this field will catch it right away. But if you put out WSP81f/r, you will confuse them a bit. Just a thought.

Dear Yuan,

thank you very much. I have already  proceed the 'experimental' samples (set up: Samples + positive control + negative control). but I have got band in positive control and there is no band with samples so we can play in pcr cycling or what should I do?

regards,

Hi Samia,

There can be many reasons causing your samples to have no PCR band. Let's trouble-shoot one by one. Let's check the DNA purity first. If your DNA is not clean enough, possible PCR inhibitors can present in your DNA samples.

1. Is your positive control a 'plasmid' or 'genomic DNA'?

2. Could you tell me how did you get your sample genomic DNA? Did you isolate them? Did you isolate them with a kit? What are their purity?

3. You can find out their purity by measuring the ratio of OD260/OD280 (see attachment)

"Nucleic acids and proteins have absorbance maxima at 260 and 280 nm, respectively.  Historically, the ratio of absorbances at these wavelengths has been used as a measure of purity in both nucleic acid and protein extractions.   A ratio of ~1.8 is generally accepted as “pure” for DNA"

Dear Yuan,

I will check for that as you said may be many reasons causing my samples.

please find the attached file. I used RRS Gene to my samples to detect the  Rickettsiella and I used ikb which the lenth of amplified sequence is 1,538bp.

and Ta is 52c and I have got this result.

thank you very much

Dear Yuan,

Another thing I would like to ask you about DNA purity?

I used picogreen to for measuring nucleic acid concentration but I don't about NanoDrop spectrophotometer if it same idea or not.

thanks

Hi Samia,

1. Right, there were no band in the picture. The very faint band at the bottom of the gel are primers. That is normal for a gel.

2. Do you have a NonoDropTM spectrophotometer in the lab? If not, you can use any spectrophotometer to measure your DNA's OD260 and OD280, and calculate the ratio. NonoDropTM is a modern-day spectrophotometer, and it is very convenient, without needing a cuvette and need only a drop (~ 1uL-2 uL) of your DNA sample.

3. The concept of them are somewhat different: Picogreen (dye) is used with NanoDrop Fluorospectrometer to precisely quantify the double-stranded DNA (without single-stranded DNA or contaminants, such as proteins) (see attachment). 

For DNA purity measure, if you have a NanoDrop, it is very simple, just select OD260/OD280 mode on the program, and drop one drop of your sample on its pad, that is it. You will get the ratio right away. No dye or anything else needed. OD260 measures all nucleic acid (dsDNA, ssDNA, RNA), OD280 measure contaminants such as proteins (see attachment #2).

hello Yuan,

how are you?

can you explain to me what does it mean Cytoplasmic incompatibility if you know?

because i did not understand it very well.

many thanks

regards,

samia

Hi Samia,

Did you mean for plants or insects (are you dealing with insects?)?

Hello Yuan,

Do you know how can i prepare10% of  potassium hydroxide?

thank you very much.

samia

Hi Samia,

For that, you can take 10 grams of KOH pellets and add it into 100 mL water; the solution will be 10% KOH. You can scale it down too, for example 5 grams in 50 mL water.

I also found this article online. See page 9 for preparation of 10% KOH.