Hello, you can marge two or more isolated DNA solutions and then use isopropanol precipitation and centrifugation at 10000 or you can use vacuum dryer or DNA vacuum concentrator for drying DNA samples. You can use two extraction dry them and use appropriate amount of TE or other diluent for making new solution with high concentration.

Good luck!

If you perform two separate DNA extractions but when you get to the elution stage, you put 50 uL elution buffer in the first tube and centrifuge. After centrifugation or re-hydration put the dissolved DNA of the first tube to the second tube. By this you can increase the concentration of DNA by about 50 to 70%. Alternatively, you could precipitate the DNA with ethanol or polyethylene glycol and re-suspend in a smaller volume.

Hello, you can marge two or more isolated DNA solutions and then use isopropanol precipitation and centrifugation at 10000 or you can use vacuum dryer or DNA vacuum concentrator for drying DNA samples. You can use two extraction dry them and use appropriate amount of TE or other diluent for making new solution with high concentration.

Good luck!

In this case, I want to know that whether you have quantified the PCR product or the eluted PCR product of second round?

If you want to increase the PCR yield, a number of factors can be examined:

https://www.neb.com/protocols/2012/06/01/guidelines-for-pcr-optimization-with-phusion-high-fidelity-dna-polymerase

Other Links

for general PCR reaction

https://international.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide

For taq PCR Kits

https://international.neb.com/tools-and-resources/troubleshooting-guides/taq-pcr-kit-troubleshooting-guide

If you are eluting PCR product of second round from gel, then you can go with as suggested by Karen and Maurice.

At last I want to mention that mostly, Gel Extraction Kits result in low yield but highly purified DNA. If your objectives are gene cloning and expression using the desired PCR product, you need not to worry. The amount you have after elution (10 to 15 ng/ul ) is sufficient for such studies.

Thanks

pcr product is very stable.Just put it in a tube on a heating block at 80c and let it evaporate for a while if the salt concentration increasing does not matter. Better to run a few more cycles of pcr if you want more product

Dear Lumayno

I think the more better is to increase number of cycles but in counted steps. No sharp increase. The high DNA concentrated samples may be diluted to achieve the work instead of re-extraction of concentrated DNA. More elation buffer from 50-1-- micro liter may be demand in some cases. Regard

Thank you everyone for your inputs! I’ll try running a few more cycles.

my concern, I am using a fastgene gel/pcr ectraction Kit for dna purification from the PCR product. this kit uses column and doesnt give me a precipitate (pellet).

Regarding the Isopropanol precipitation (Not so familar with this) is this protocol ok?

for 100uL PCR product

1. Add 20ul of 3M NaOAc

2. Add 80ul of Isopropanol

3. Place in -80degree Celsius freezer for atleast 15minutes

4. Centrifuge at max speed for 20minutes

5. Wash pellets with 70% ethanol (dna is 270 +bases in size) (or should I use 95% ethonol for this?)

6. Resuspend in UPW

Another concern, I am working with 6 genes, three of which showed 1 band each in gel electrophoresis (they are my target sizes) while three of which showed 1 strong band (my target size) and few weak bands.

I can try the Isopropanol precipitation for the three genes showing 1 band only but for the other three, I have to run these in TAE gel for me to isolate the target band. I don’t know if Isopropanol precipitation will work.

To Dr Phulara, I have used the eluted DNA (these are the extracted DNA already; not sure if you meant the same, I apologize for that). To run another round of RACE PCR.

Ususally Extraction kits give low concentration of DNA. Better to use Trizol method for extraction, It may give high concentration of DNA.

BTW whats your sample?

Sanny,

In addition to optimizing the PCR conditions (running more cycles and playing with conditions for the genes which don't give a single band) try to elute the DNA products from the same column twice into the same tube by using 20ul of the elution buffer each time = 40 ul final volume. If that approach won't increase the DNA concentration, do the opposite: use 50 ul twice, then partially evaporate the buffer as Paul suggested earlier.

Also, make sure your blank solution for the spec or a nanodrop is the same as the elution buffer. Good luck!

Thank you for your replies!

Sahid, I am working with fish brain samples to purify genes (gnrhs, gnrhs and kiss) for the real-time PCR (standard preparation).

Dr Irina,

I’ll take note of that. Thank you!

No problem! It would be helpful to know what actually worked for you. Can you please post an update later? Thank you, Sanny.

Hello everyone! Thank you all for your suggestions!

I have tried some of your suggestions but I guess the best one is through ISOPROPANOL PRECIPITATION.

I added 1 ul of Glycogen (20mg/ml) as a Carrier to make my pellets more visible.

I worked with 200ul PCR Product. I eluted the pellets with 10ul of Elution buffer (prewarmed to 70degree Celcius).

I got 190 to 240 ng/ul of DNA Concentration.

using the Fastgene kit, I eluted with 6ul of prewarmed Elution Buffer and only got around 100 ng/ul of DNA concentration. (I reuluted the column again with 10ul and got around 30ng/ul of dna concentration).

Now, I am trying to find a protocol to extract DNA from TAE agarose gel without using any kits. (since three of six genes that I am working are showing a few weak bands at electrophoresis). I have tried using the geneclean (dissolving gel with NaI and addition of glassmilk), however when I check for the DNA concentration, the ratio (260mm/280mm) is too high (30-80)! most probably it's contaminated.

Increase the number of amplification cycles.

Increase template concentration.

Try a different polymerase.

Try dNTPs of a different origin (dUTP arising from deamination of dGTP inhibits amplification by proofreading polymerases)

Optimize dNTP and Mg2+ concentration.

Redesign the primers.

You can always use a speed-vac to concentrate your samples by evaporating the water.

If you have tried Increasing the amount of template DNA and number of cycles and still the product is less.

Try adding glycogen while precipitating DNA with sodium acetate and ethanol, to enhance the yield.

In case you have some non-specific amplifications, you can try electroelution of your expected PCR amplified product and you can add glycogen in this procedure as well to precipitate the DNA after electroelution. In this process, you recover >90% of DNA, whereas using kits reduces the yield a lot.

Good Luck!