Dear Gemantari,

I'm sorry but I couldn't see your band. What is the size of your amplicon?

1. Even if you have not quantified using nanodrop or other spectrometric quatification methods, you can always check it by agarose gel electrophoresis. Before using for PCR, you should always check the quality and quantity (can be approximately estimated on an agarose gel).

2. Moreover, for cloning purposes, you should first use a small scale PCR (50uL reaction) and check whether it has worked or not. Further when you need to amplify the insert in bulk, you can use the previously amplified PCR product as template. That will ensure more specificity and will have enriched template.

3. When you have purified your sample DNA, I think you could have used a lesser volume to obtain more concentrated sample. May be your template is too less in that 2uL. You can either use higher volume of the same elute or purify fresh in a lesser volume next time.

4. Also, make sure your PCR reagents are fine and PCR is indeed working. Set up one no template control (negative) and one positive control standardized and known to work under same PCR conditions. Use fresh primer dilutions because multiple freeze thaw of oligos sometimes leads to degradation and low amplification in PCR.

Hope it helps!

Dear Gemantari,

I'm sorry but I couldn't see your band. What is the size of your amplicon?

1. Even if you have not quantified using nanodrop or other spectrometric quatification methods, you can always check it by agarose gel electrophoresis. Before using for PCR, you should always check the quality and quantity (can be approximately estimated on an agarose gel).

2. Moreover, for cloning purposes, you should first use a small scale PCR (50uL reaction) and check whether it has worked or not. Further when you need to amplify the insert in bulk, you can use the previously amplified PCR product as template. That will ensure more specificity and will have enriched template.

3. When you have purified your sample DNA, I think you could have used a lesser volume to obtain more concentrated sample. May be your template is too less in that 2uL. You can either use higher volume of the same elute or purify fresh in a lesser volume next time.

4. Also, make sure your PCR reagents are fine and PCR is indeed working. Set up one no template control (negative) and one positive control standardized and known to work under same PCR conditions. Use fresh primer dilutions because multiple freeze thaw of oligos sometimes leads to degradation and low amplification in PCR.

Hope it helps!

Dear Baiq Maylinda Gemantari,

Yes, you can when you have established a protocol that cant vary or not be influenced  on dna concentration, for this you have to try with different DNA concentration in pcr reactions and need to analyze the pcr band. So, from that observation you can conclude to do pcr without measuring the DNA.

But For some sample you may need to do that. Sometimes protein present in the extracting DNA can causes a great problems so for those samples you need to take measurement for DNA.

Good luck !

dear Gemantari,

Though, you already purifying your DNA template for PCR, it is not guarantee your PCR reaction will success. did you know that for PCR needing around 20-50 ng DNA per reaction? This is why quantify DNA concentration first is a good idea, so you know how many micro L you should take.

Honestly, I barely see your band in that photo. are those have correct length as you wish? 

Good Luck!

Dilin

Dear Rabbi, thankyou so much for your suggestion.

Dear Saha and Nataningtyas, I am so glad to have your answer here. I am doing my thesis about primer optimization, honestly to get an optimum annealing temperature to amplificate DNA human. On that pict, the first lane is ladder and next is PCR product which has different annealing temperatur. I used 25uL reaction for each tube I amplified. Length I expect is 115bp that has already showed by the second lane.

Dear Kumar, the amplicon size is about 115bp. Marker I used was 50 bp. The first bottom one of marker you said is 50 bp, second is 100bp and third is 150 bp. I am sorry I didnt give the details about pict I attached. Do you have some suggestion to make my band brighter?

Dear Baiq Maylinda Gemantar,

In my lab i am doing routine pcr without measuring DNA concentration, when everything is okay. As because from a prolong  observation i have found that DNA concentration vary from 40-250 ng/ul so i have no problem with that when i am using 2ul Temple DNA in a 25ul pcr reaction. But i am using Taq master mix that can work from 50-1000 ng/ul per reaction.

The gel photo you have given, have some clues that can help you to analyze some problems .

1. The DNA concentration you used is normal as because if DNA concentration is so high then you will see smear in the Gel photo. So, he that is not much smear.

2. Problems can be happened in annealing temperature and extension. For a perfect annealing you can run a gradient pcr around your primers annealing temperate from where you can have a perfect annealing temperature.

3. Are you using ready made Master mix or your have made you pcr master mix with Taq, dntps, Mgcl2 etc. ?, if you made your master mix manually then may be you have added less dNTPs for this enough pcr couldn't yield perfectly.

4. For annealing temperature, 1000 bp/1 minutes, so 30 sec is enough for your pcr product. sometimes 45 sec can give better yield.

Can you share your pcr master mix composition and thermal cycling condition ?

So hope you will get sharp band and good yield in pcr.

Good Luck !!! :-)

Dear Baiq Maylinda Gemantar,

1. I did see your faint PCR bands on the gel with correct position after you provided more info.

2. It is useful to quantifying your DNA samples, so you will have an idea how much DNA template you used. Besides, this data might be needed in the future to write in the Materials and Method section if you try to publish a paper. While quantifying your DNA samples, you can also measure its OD260/OD280 ratio to find out your DNA quality ("purity").

3. You should be aware that even you have purified your DNA and use proper amount of them for PCR, you sometimes may still encounter no-PCR-results problem. For example, if your DNA template is GC-rich, it may hard to be amplified. In this case, special PCR additives (such as DMSO, and others) can be added to improve PCR accuracy and efficiency. Commercial 'for GC-rich temperate' buffer can also be used.

4. Your DNA product is short (115 bp). So, you will expect that the 'brightness' of your DNA bands will be less than that from longer DNA products if you have same cycles of PCR amplification. This is because of that the long DNA product 'harvest' and accumulate more EtBr when run on the gel.

5. If you want more sample of your PCR, you can gel-extract the band on the gel and use it to re-amplify again (use hi-fi PCR polymerase to prevent mutation). Remember to sequence the PCR product afterword before you use it for downstream experiment.

Some things to remember..

first you have to select the annealing tm and select the amplicon size

Check the quality and concentration of dna ,A  single thick band is our expectation in agarose gel.

By seeing your gel pic the problem may be in your dna, primer, or in your master mix.

If you are taking too much con of dna that may cause the formation of dimers or false products but at the same time we can select our expected product size but little difference may occur in its size....

Hi,

I am not sure what exactly your question is. Are you asking if it is crucial to quantify, i.e. measure gDNA (template) concentrations before performing PCR? In my experience, there are many concentrations that matter (dnTPS, magnesium, primers, annealing temp. etc.), but DNA template concentration does not seem to have much of an effect. I usually perform PCR on hundreds of samples at once, and in most cases I skip the step of measuring DNA template concentrations (i.e. Nanodrop). I have successfully amplified various loci in animals and plants with DNA template concentrations ranging from 5 ng/ul to 200 ng/ul. So my piece of advise is: I would not worry so much about the template DNA quantity. Before you quantify and vary your template DNA concentrations, I would first vary primer concentrations, annealing temperature, number of cycles, etc.  in order to improve your PCR results.

(Looking at your gel picture: I do see some faint bands in the first two wells that appear to be amplicons, but without knowing the size of the ladder you used and the size of the targeted amplicon, it's hard to say whether this was a success. I do not see any primer dimers, which is a good sign--you might want to increase primer concentrations as a first step.)

I hope this helps.

Cheers, Oliver

I'd check your primer concentrations. Normally, the primers show up as a big blob at the bottom of the gel (unless you're staining with SYBR-green or another double-strand specific dye). Nothing of that kind visible on your gel, which makes me think your primer concentration is too low. 

As far as template DNA concentration is concerned, this isn't an issue. A smear of chromosomal DNA is visible just below the wells, and if you've got enough DNA to see on a gel, you've got enough to give you a PCR product. 

So it's the efficiency of your PCR reaction that's the problem. 

I am agree with Andrew Jenkins specially for this words .....

As far as template DNA concentration is concerned, this isn't an issue. A smear of chromosomal DNA is visible just below the wells, and if you've got enough DNA to see on a gel, you've got enough to give you a PCR product.

Has anyone ever experience with PCR without quantifying DNA template? - ResearchGate. Available from: https://www.researchgate.net/post/has_anyone_ever_experience_with_PCR_without_quantifying_DNA_template [accessed Apr 27, 2016].