Two things may lead to loss of your DNA. First (and most likely) is that you have over dried the beads. The weak electrostatic interaction of DNA/beads becomes strong hydrophobic interaction. Do not dry for more than 1 min after the 70% wash. Second reason is that you might have the opposite - if there is residual ethanol (5-10%) than DNA won't elute  well. The nano drop result is due to PEG in the AmPure - do not worry too much about that. The last purification is absolutely necessary for NGS , because you will have to remove the adaptor and primer dimers. Hope that helps.

Two things may lead to loss of your DNA. First (and most likely) is that you have over dried the beads. The weak electrostatic interaction of DNA/beads becomes strong hydrophobic interaction. Do not dry for more than 1 min after the 70% wash. Second reason is that you might have the opposite - if there is residual ethanol (5-10%) than DNA won't elute  well. The nano drop result is due to PEG in the AmPure - do not worry too much about that. The last purification is absolutely necessary for NGS , because you will have to remove the adaptor and primer dimers. Hope that helps.

I totally agree with Dr. Petre Lanakiev, as he mentioned ethanol plays major role here,

some times practical difficulty using beads wash plays major role, Confirm your 70% Ethanol preparation is on the day of experiment not the older one. it should be exactly 70% (70 ml ethanol + 30ml DDW) 

Do not dry for long time as petre suggested.

If you feel its missing in the beads, you can reelute the products from the beads after the first elution, i have done it so, i got some good amount of Product, so you can try this.

ALL the best.

Illumina's protocols call for 2 serial washes with 80% ethanol, freshly made. I read somewhere that the use of "80%" is in case the stock alcohol bottle has absorbed water from the atmosphere and is not actually 100%.  The idea was that if the stock bottle is off, at "70%" there could be too much water present and that your DNA could dissolve and be washed away.  I've found that if I use 3 serial 80% washes, my DNA is retained and I don't have salt problems in the Blue Pippin. ( N.B. I deliberately leave a little excess behind in the first 2 washes to minimize bead loss with my magnet. ) At 2 serial 80%washes, the retained salt was problematic for the Blue Pippin.

Ratio of Beads to DNA volume is critical, Also re-suspend the beads well before adding to DNA. In case if you have used different amount of beads unknowingly you will loose DNA. 

Thanks very much for all your help. I used the 1 minute for drying beads, that kinds like got better. I run 2% gel for checking ( the size of the DNA is around 150bp after PCR). The NEB protocol required 80% ethanol wach twice and dry for 10 minutes. 10 minutes should be too long for drying beads. I will try wash 3 times to see if the DNA would be more pure. So anybody have idea why I could got 90% of DNA yields with beads wash before PCR? But only collect so little yields with beads wash after PCR? The NEB tech support said 1ug of input DNA will provide about 500ng yields. Does that enough for the NGS?

500 ng should be plenty for NGS provided that your size is correct. I will double check the size, it seems too low unless this is smallRNA library. It is imperative to quantify by qPCR before committing to NGS sequencing.

For the PCR effect on AmPure I have seen similar effect when I over amplify the library. Stick to low cycle number and add >5min final extension. Reason is that during the PCR you might get 1) higher order structures and/or ssDNA fragments, neither is very good for AmPure (too sticky) 

Good luck,

Peter

Dr. Lanakiev, Thanks for your response. Do you recommend 1 DNA : 1 volumn BEADS ( NEB protocol) or 1 DNA : 1.8 volumn of Beads for the last step clean after PCR reaction? Thanks again.

I will suggest that you use your vendor protocol if they provide the beads. Most vendors will buy AmPure beads and reformulate the buffer they are suspended in. That being said if you are using Beckman AmPure 1.8 to 1 is the ratio that will get rid of anything below 150bp. What exactly is your application? Sometime when you are doing siRNA there is double size selection, when you are using 1:1 (or even less) and then you are adding more beads to make it to 1.8 x  to eliminate both smaller and bigger than desired fragments. 

Hi, Dr. Lanankiev: I am trying to build up a DNA library for the sequencing. The starting material is cDNA . The Ampue beads I purchased from beckman directly. I have an idea: If I can not get the enough amout of PCR product after clean up by beads. I collect that clean PCR product to run the 2nd time PCR. Do I need to clean up that 2nd time PCR product again or that can be used for sequencing directly? If Nanodrop is untrustable for the DNA cleaned by beads. How can I sure about the quantification of the PCR product for sequencing? Thanks very much. 

If this is an RNAseq project, you are definitely over amplifying the library (you indicated 15 ug after the PCR). I will be very cautious not to use more that absolute minimum necessary PCR cycles. If you are looking for expression levels all these will be messed up  by the PCR bias.

At this point in my opinion you will be better off starting the LC from the beginning, if you have enough RNA. 

You need very little DNA for sequencing ( 2nM Library  stock is further diluted to 15-20 pM before going into the flow cell for clustering). 

P.S. The Library concentration must be based on qPCR before going into sequencing. You will also need precise DNA sizing for the library to come up with the qPCR estimate. KappaBiosystems kit is the golden standard for QPCR for illumina libraries. 

Hi Pradeepa,

what did you mean by re-suspend the beads well before adding to DNA ?

thanks

samia

Samia,

store beads in fridge (4oC) between uses to minimize growth of bacteria, mold or other potential contaminants.

The suspension of beads in PEG & salt need to be at room temp and well mixed for them to work properly, so before using allow the beads to sit at room temp 10-20 minutes.

The beads will settle out of solution, so before adding them to your DNA always be sure to mix them WELL by shaking or vortexing.

The volume ratio of beads to sample can be altered to recover different size ranges of nucleic acids. Basically, the higher the concentration of PEG & salt (that the beads are suspended in) the smaller sizes of nucleic acids will precipitate and bind to the beads. Ampure, Kapa & Zymo now all have published size specific cut-off ratios. Look them up and think about what it is your are trying to clean up from your samples to get the optimal product.

Best of luck!

Selene.