Lazy way: You can repeat your PCR as many time as you want, add up all the tubes together, purify the whole on a kit-column and evaporate some water out of it using a SpeedVac. I recon it is a lazy way, but sometimes you just want the sequence, right ?

Run a PCR once again using the amplicon

Whats the model and brand of your automated sequencer ?

Another possibility would be cloning the PCR product. If you used Taq polymerase you can clone it with Topo TA and sequence from the plasmid after mini prep. If you used a proofreading polymerase you can clone it blunt end into any vector.

Hi Lavender,

As Gabriele said, exactly the same question arose in my mind.

As my opinion if you are doing gradient and your band is single but light.then you can use the hot star master mix of Quigen on the place of 10x buffer and Mgcl2, Taq gold and dNTP etc it will give you good result as compare to make a mix.Another thing what is your final volume of PCR product ? if your final volume is 25 and you are using applied biosysytem sequencer for sequencing then for sequencing PCR it need only 5 ul of you pcr product, so when you have to do your purification then put as less as the elution buffer, because of this you can gt more concentrated product.

Good luck

I am with Gabriele

and I would add DMSO to the reaction, sometimes helps opening DNA with difficult sequence or very long PCR.

second, if you are able to see your bands in a gel (0.8% - 2% agarose) you have enough PCR to sequence is a matter to send enough volume to be sequenced (after purification or exo-saping primers) ...

Agree about the cloning! Need very little PCR product to lone into a TA cloning vector (i. e TOPO), and will probably improve your sequencing results.

Run gradient PCR with the same amount of samples and reagents, vary the annealing temperature. If the amplifications are consistent, u should all the samples amplified at the size you are interested in. Choose two or three bands and purify the PCR products using one purification process.

I think you should repeat your PCR and try to optimise the settings (temp and number of cycles) but if you need only to concentrate DNA/RNA in solution here is an excellent protocol:

Concentration of DNA or RNA in aqueous solutions

This is a method to concentrate DNA or RNA in solution by water uptake by dry-butanol. Butanol will take approximately 20% of its volume in water from the DNA/RNA solution.

1. Add butanol to remove 10-20% (usually 20%) of its volume e.g. 100ul to remove 20ul from the solution.

2. Mix and spin down full speed for 1 minute

3. Discard butanol phase

4. Add 500 ul of H2O-saturated diethylether

5. Mix well and spin down

6. Discard supernatant (diethylether)

7. Repeat points 4-6 2x

8. Heat up the samples at 37oC for 5 minutes

if you have enough DNA in your PCR but just the wrong concentration, you can put the tube on a termalcycler at max 65°C and than resuspend it in the correct volume or precipitate it (NaAc+ethanol) if the volume is more than 15 ul (DNA would stick to the tube wall).

But keep in mind that in this way you may have problems in sequencing due to contaminats concentration (eg. buffer salt).

I think you should try re-amplification of PCR product with the same primers and same condition. this will amplify the amplified product and concentrate it.

if your light band is 3000 bp, I would be satisfy. The best thing is to clone it.

Anyway run a new PCR with the aplicon obtained as template. If you want to obtain 1ug of your band of 3kbp you should calculate how much primers you need in molar quantity. Sometime PCR reactions are set up for diagnostic purpose not for producing high quantity of DNA

you could concentrate PCR products if you have access to a speedivac

Or try using a 2nd Round of PCR with internal primers to those you have and using the PCR product as a template but use a dilution say 1 in 100 or 1in 50

In the presence of some problems with the amplification of the target gene (e.g. weak bands or non-specific secondary bands after the PCR reaction) which hinder the assessment of the PCR product and further analysis, a gradient PCR technique should be carried out, which allowed the empirical determination of an optimal annealing temperature. This can be achieved by choosing a temperature range (e.g. 50, 52, 56, 58, and 60° C), and setting this across different columns of wells (at different annealing temperatures) in the thermocycler. For example, column 1 for 50° C, column 2 for 52° C, and column 3 for 56° C (i.e. gradually raising the annealing temperature in order to amplify the target gene). With each sample, the PCR mix had the same concentrations for all the reactants. Thus, each reaction will be performed at a different annealing temperature, with all other values being the same. PCR is normally started at 5° C below the calculated (referenced) temperature of the primer melting point (Tm). Theoretically, the annealing temperature (TA) = (Tm) - 5°C. The melting point (Tm) can also be calculated using the following equation: Tm (°C) = 4 (no. of G + C) + 2 (no. of A + T)

I agree whith John Damiano.The easy way it is to put inthe speed vaccum.

I do not know the lamp procedure, but you always can purifie your band from an agarose gel. Another tip, adding more template DNA in PCR do not improves the final yield of product, some times is the other way round and adding 10 times less DNA input you obtain better results (becouse you are diluting the contaminants in the PCR). The yield of final product can increase adding 10 % glicerol to the PCR mix or 10% of DMSO if your fragment is GC-rich, or adding albumin.

You can concentrate DNA using microcons (Millipore) is easy, fast, high recovery and do not concentrate the amount of salts in the sample.

I use the method described by the MoBio kit:

When the final volume of eluted DNA will be 100 ul. The DNA may be concentrated by adding 4 ul of 5 M NaCl

and inverting 3-5 times to mix. Next, add 200 ul of 100% cold ethanol and invert 3-5 times to mix.

Centrifuge at 10,000 x g for 5 minutes at room temperature. Decant all liquid. Remove residual ethanol in

a speed vac, dessicator, or air dry. Resuspend precipitated DNA in sterile water or sterile 10 mM Tris.

I would also recommend to find out the optimal annealing temperature for your primers as Fahd described before. A further step to increase the yield of your PCR product would be the use of touchdown PCR method. A final word, do not add too much DNA into your PCR. Dilute your DNA, do some serial dilutions of your DNA, e.g. 1:5 or 1:10.

MinElute by Qiagen works well for purification and concentration of amplicons less than 4kb. Try to pool as much of your PCR product as possible and run it through MinElute. The elution volume is typically 10ul.