The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A ratio of ~1.8 is generally accepted as “pure” for DNA (see attached NanodropTM bulletin). If you are using a ABI DNA sequencer, the recommended DNA quantity depends on the size of your PCR product. For 500-1000 bp, 10-20 ng is needed (see attached ABI PRISM BigDye Primer booklet, page 13, Table 3). So, I think you are good to go. Try and find out.

The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A ratio of ~1.8 is generally accepted as “pure” for DNA (see attached NanodropTM bulletin). If you are using a ABI DNA sequencer, the recommended DNA quantity depends on the size of your PCR product. For 500-1000 bp, 10-20 ng is needed (see attached ABI PRISM BigDye Primer booklet, page 13, Table 3). So, I think you are good to go. Try and find out.

A ration A260/A280 is generally good is it is between 1.8-2.2. The concentration needed depends of the fragment's size. And the volume required is generally 10-12 microliter per sequence.

But, you should verify directly with the sequencing unity.

Generally it is better to do a "TA cloning" and a transformation to have enough plasmide and the beginning of the sequence don't have a good resolution (between 10 to 100 bp will be impossible to use).

If you dont want to use plasmid DNA for sequencing, Alternatively you can load  your PCR product on gel and do gel extraction. Following this you will remove all result interfering contents like free dNTPs, unused DNA template/ primers etc. Ideally >= 500ng of this PCR product will give good length of read.

Sequencing PCR fragments is normally fine, but you have to be sure that you have amplified a single specific band in your reaction. If this is true, as you correctly say, it will be sufficient to remove primers and dNTPs, either by a PCR purification column or by a digestion approach (tipically with exoSAP - exonuclease I and shrimp alkaline phosphatase) followed by a phenol/choloroform purification and ethanol precipitation. If you have unspecific bands in your amplified product you should either gel purify or TA clone as suggested by Laure, selecting the clones containing the insert of the correct size for your specific product.

For the rest, your 260/280 nm ratio is slightly high for DNA and it could be due to a product rich in adenosine or it could mean that you still have quite some free dNTPs in the mix (adenosine 260/280 ratio is >4 and will therefore shift your global ratio higher proportionally to its amount: check the technical bulletin Yuan-Yeu has attached for you).

Concerning the right amount of DNA product and primers to use you will have to see with your sequencing company or service, since there is quite a widespread range of suggestions. My personal rule of thumb is to use about 5-10 ng every 100 bp of PCR product, with 3.5 pmoles of primer (with this amounts and ratio I can read 8-900 bases highly reliably).

Good luck.

PCR product to sequencing can be done easily as above people stated clearly, Do either gel load and purification, or do pcr production purification kit from many manufacturers (We use Qiagen)  for sequencing ur protocol matters, you will be need only 5-10microlitr of PCR product only, which should be exclusive of Pcr contaminants such as enzyme, dntp, primer dimers etc.,

So purification of PCR product for ABI dna sequencing is best way. No need to quantify your DNA concentration using any method, if you could visualize it in the 2%gel, which means your product is above 20ng, which is morethan sufficient for sequencing.

Best wishes

nice readings. go ahead for sequencing. 

It is really important to remove the NTPs and primers from your PCR before sequencing unless you design your reaction to use up these reagents.  Have you purified your PCR product?  If not then your OD readings are not valid

You are absolutely right Martin Pentony before preceding to sequencing a researcher should purify/ clean the PCR product. If not till that OD should not take in to the consideration for the rest procedure.

In General considerations:

Geleta Dugassa Barka should have an idea to run the PCR- the amount of DNTP mix added and for e.g. last cycle is 30 or whatever "what would be the estimated conc. of DNTP left over.?? "  if you are not sure or sure then also purification is needed.  

PCR cycle and the building blocks (DNTPs, Frwd primer, reverse primer etc..) is following linearity till the sufficient quantity is supplied. Make sure during PCR it should be supplied building blocks are well exhausted and if remains in trace amount then it must be cleaned out and then may proceed for sequencing.....

Good luck for the rest!!!

Thank you all for sharing your invaluable experiences. An other problem in using PCR product for sequencing is the high concentration of DNA. I know that the con. of plasmid DNA with insert should be about 100ng/microlitre but Iḿ quite surprised to find out on some literatures that PCR product DNA to be used for sequencing is far less than this conc., is about 1pmol/microlitre for a 100bp segment of DNA (target DNA to be sequenced). Still am not sure the best conc. of SP6 and T7 primers as a con. of 0.8microM resulted bad sequence (with

The ration 260/280 is around 2 is a good measure. I am sure that you should also test the RIN (using Bioanalyzer), which should be between 9-10 RIN. This number gives you an indication about the degree of fragmentation of ur RNA. 

@ Geleta,

I am confused about your question at beginning (using PCR product for direct sequencing) and then the last message (SP6/T7 primer con. needed) you posted:

1. You can only use SP6 or T7 primer as sequencing primer only when you have cloned your PCR fragment into the plasmid (which has this SP6 or T7 site).

2. If you are using 'PCR product' for direct sequencing, you must design and use gene (or your DNA fragment)-specific primers for sequencing.

3. You cannot use SP6 or T7 primers as the sequencing primers for 'PCR product'-direct sequencing.

Which sequencing method are you currently using?

@Yuan-Yeu Yau

I'm grateful in your effort to help me. I'm working on different expression libraries of differentially expressed genes (more than 200). Each clone is EST which are previosely cloned by plasmid vector (pGEM-T easy vector). The concentration of most of my plasmid DNA with ' insert'  is less than 100ng/microlitre. I used SP6 & T7 primers to verify whether the extracted plasmids are with insert. Using the same primers (SP6 & T7), concentration being reduced from 2micro molar to just 0.8 micromolar, I tried to sequence my inserts. But unfortunately the capillary raw data read of the sequencing machine signals indicate my sequence reads are with in the NEGATIVE BASELINE READ which inturn found to be very bad quality sequences (less than 1% quality, as seen using chromatogram map). I'm using AB Biosystems 3130/Gene Analyzer sequencing machine. Currently am trying by changing the conc. of my PCR products and primers but as to yet no any breakthroughs. I'm hearing to any assistance. txs.

1. I see, So basically the fragments were already cloned into pGEM-T, and you were using the SP6 and T7 primers to amplify (to check) if any fragment in the plasmid. Once they are amplified, the PCR product ends will now contain SP6 and T7 site and can be used for direct PCR fragment sequencing using SP6 and T7 as sequencing primer. That makes sense.

2. In that case, why don't you just use the plasmids (detected positive with an insert in it) for sequencing? Usually people do PCR product direct sequencing is to avoid the cloning step. In my experience, the plasmid template gave me better sequencing results. I know you are worry about the concentration, but why not just go ahead and sequence a couple of them to find out the results. Maybe it will give you good results. You never know.

3. Besides, when you give a 'concentration (100 ng/microliftre)' of DNA to an audience, it will be helpful to provide a little more info to it--such as 'volume'. A tube (stock) with 1 ml DNA solution of 100 ng/microliter or a tube with 5 ul DNA solution of 100 ng/microliter can make a difference in how many downstream experiments you can do. The former one can always be concentrated (such as using a freezing SpeedVac) to a much higher concentration if you need it.

Hi. I have thought that we cant use PCR product for determine its concentration directly with use of nanodrop. but anyway, i usually send my PCR product for sequencing directly (2 reads)  and company purify it and i did not have problem yet.