disappearing band

[frg]

Hi everyone

I amplified a PCR product and got a nice band of the correct size on a gel. I then gel extracted the PCR product and ran a sample on a gel and got no band whatsoever. This was despite a nanodrop estimation telling me i had 100 ng/ul of DNA in my sample.

I then re-gel extracted some more of my original DNA prep and used the nanodrop to estimate the DNA concentration and it was about 90 ng/ul but i noticed the 260/280 ratio was 4.49.

What does this indicate? Does it mean my sample is contaminated by nucleases and is potentially degraded hence why i cant see it on a gel? If so would the likely source be the water i am using to elute the DNA after extraction?

Any thoughts would be greatly appreciated

[relaxin]

It just means that the concentration of the eluted DNA is too low to get anything meaningful. You can reamplify the PCR fragment using 1 ul of the eluted DNA.

[Vik]

Couple of questions
1. What size band are you isolating and what method of isolation are you using?
2. You got an OD of 4.49??? Are you sure that it is not 1.49? If so, I agree with relaxin that you probably have no DNA present in your extracted solution.
Nuclease degradation is possible but pretty rare. As a rule you dont have many DNases running around a lab. RNases though.... *S*
Try changing your water for sure. I assume you are using sterile, or DEPC treated water for elution?

Vik

[mchlbrmn]

I don't think it's common to use DEPC treated water for DNA (and some now don't recommend it for RNA). As mentioned, DNAse contamination is rare.
90ng/ul in a Nanodrop is not a low reading. If it's not DNA, and the ratio is odd, then it sounds like there's contamination of something else. Are you sure you used a normal elution solution, such as water or low concentration Tris , maybe with EDTA? The negative yield, and then positive yield with a bad ratio suggests some kind of contamination. Consider the other sources of contamination, such as all the solutions you used, and whether they were properly washed away, and removed from the column.

Since it's a Nanodrop, you can look at the curve and see where there's an unusual peak, and sometimes that can help diagnose it. Phenol absorbs about 270, I think (not the case now). THe Qiagen gel dissolving reagent absorbs way to the left, but you shouldn't have that for a PCR clean up. That one often is often not completely removed and can raise the 260/280. Oh, I just checked and this is a gel purification: as I said it's not uncommon to have a low absorbance contaminant peak leftover, and for low DNA yields it' becomes relatively big. I see a higher 260/280 ratio for gel pure samples, but not that high. But, if you use a different kit or had a technical issue removing the solutions the result may be different. It's also important that the agarose be completely dissolved. If not, I can imagine undissolved agar harboring these contaminants and affecting the OD. The final spin of the column after removing the flowthrough (qiagen protocol) is probably important to remove the dirty wash solution.

If you get a single clean band on the gel, you can use a PCR clean up column instead of the gel purification, and this is a more reliable method.

[sean.laub]

Not sure if you're able to quantify PCR products with nanodrop. I don't know why this is, I remember reading this somewhere.

Recently I performed PCR and gel extracted the relevant band using a gel extraction kit before reading it on the nanodrop. It gave weird results.

I then ran the fragment on an agarose gel and quantified it alongside a marker (by judging the band intensity - look at the booklet that comes with your marker).

I should also mention that gel extraction does not in anyway give full recovery. Did you run the entire volume of the PCR into a single well and extract this? Are you sure you loaded enough of your extracted DNA into the gel? What I typically do is run a 50 ul PCR, load the whole thing into two wells (big combs) and extract this on one column. Better to have too much than not enough!!

[tsy8804]

You can try to avoid the gel (with band) exposed to long under the UV light. Because the light will denature the DNA. So, if you exposed it too long with UV, the band that you slice for purification may be lost and causing you can't get any band when you re-run the product. By the way, I am using nanodrop to check for the purity as well. But most of the time it will give me incorrect reading.

[r.rosati]

That's weird, just yesterday a friend at my lab had the same strange reading on a spectrophotometer - about 4.5 for both A260/A280 and A260/A230. She had purified a digestion, and had a concentration of about 60 ng/ul. I suspected either a contaminant, or something wrong with the blank. We re-did the blank three times, same results, so it must be a contaminant for us too. We couldn't perform a spectral scan on our small spectrophotometer, so I'd be curious to know if you did it, and what came out of it.

[talkingtree]

Can you show us your nanodrop peak? Maybe it wasnt detecting DNA products at all

[Onyourcase]

Hi everyone

I amplified a PCR product and got a nice band of the correct size on a gel. I then gel extracted the PCR product and ran a sample on a gel and got no band whatsoever. This was despite a nanodrop estimation telling me i had 100 ng/ul of DNA in my sample.

I then re-gel extracted some more of my original DNA prep and used the nanodrop to estimate the DNA concentration and it was about 90 ng/ul but i noticed the 260/280 ratio was 4.49.

What does this indicate? Does it mean my sample is contaminated by nucleases and is potentially degraded hence why i cant see it on a gel? If so would the likely source be the water i am using to elute the DNA after extraction?

Any thoughts would be greatly appreciated

What size band did you see, how bright was it and how did you thengel extract it?

[ashu330327]

Dear Sir
I have done normal pcr of target gene. I have got the expected size but intensity of band is low. My senior is advised me to clone the purified fragment in pGEMT easy vector to increase the copy number.
Is this way of doing is correct?
Any advice would be highly appreciated!!!

[mchlbrmn]

Yes, if you subclone it you can make lots of copies of it. That may take longer than other methods, and you haven't told us what your goal is? Do you want enough to make an expression vector, do you want to make a probe, do you want to sequence it?
If you can PCR a little, the quickest thing might be to pool many PCR reactions, or rePCR some of the PCR product some more cycles.
Is the template a plasmid, or cDNA, or genomic?

[ashu330327]

sir my goal is to make deletion mutant of a gene by using pK18mobsacB vector.

[relaxin]

You need not subclone the fragment into pGEMTeasy for creating deletion mutants.

If you want deletion mutants of different size, you simply amplify fragments of different length and subclone into pK18mobsacB vector. For creating deletion mutants of just a few bases, QuikChange site-directed mutagenesis (Stratagene) kit may be useful.

For detailed information, please see the following presentation:

http://www.youtube.com/watch?v=oa10dCoRbic

[ashu330327]

Dear Sir
actuall the concentration of fused product (A n B) is low.so I have cloned this fused product in pGEMT vector to increase the concentration. what is the annealing time for fusion of Two fragments in PCR overlap extension method?
Please send me the paper which have information about pK18mobsacB vector and how it is used.

Thanks