Why do i get DNA of low concentrate after purification with kit - (Aug/27/2012 )
Please, i need advice. I performed pcr with a DNA template of 10ng/ul and used 10ul (which means i used a total DNA template of 100ng in the each reaction mixture. I used the GenElute purification kit to clean the pcr product, but i got a final DNA concentration of about 17ng/ul. I am asking if this concentration is too low for sequencing? since i have to send 5ul of my sample for sequencing. Please, any advice will be highly appreciated.
Also, i stored my pcr products before purification at +4 degrees for about 1 month. Is this ok ? Can one really store pcr products of DNA at +4 degrees. ?
What could be the possible reasons for the low concentration i got ?
Try increasing PCR cycle if they are less.
You shopuld not store DNA at 40 C, It may degrade by DNases. Always store at - 20oC.
Wow...100 ng DNA? A bit too much:) I usually use 1 ng/ kb plasmid in 15 uL PCR reaction (for example if I have a plasmid of 5 kb, I use 5 ng of DNA).
In a PCR reaction after a few PCR cycles (usually 10-20 cycles) the dNTPs, primers and so on are used up. If you have such a huge concentration from the beginning, the dNTP/primers/polymerase are limiting from the first cycle. If the PCR components are limiting/insufficient for the PCR, the amplification is done with errors.
17 ng/uL is a bit too little for sequencing.
You can usually store DNA in the fridge if you are sure that you do not have DNAses in your solution i.e. in the elution buffer from the PCR clean-up kit. But, it is no problem to keep it for long in either fridge (but better in the freezer). But again, you must be absolutely sure you don't have DNAse inside.
It add to Andreea's answer, it also depends on the polymerase you used - if you used a proofreading polymerase, you can't store at 4 deg C, as the proofreading part of the polymerase can nibble away at the ends of the product.
Did you run a gel on the PCR product? I'd suggest that you got little or no amplifcation, and simply purified your template. As above, WAY too much template. If you elute in TE rather than water or Tris, then the DNA is stable at room temperature for long periods, even with DNAse present. I'd still recommend storing at -20, given a choice, however.
Thanks for your response. I would definately try to use smaller template as suggested, but i am asking if it is possible to get results with this. This is because,I had used this same pcr protocol(10ng/ul) in a 50ul reaction mixture earlier with good results after sequencing, hence the reason why i used it again. I did run my pcr product before purification on gel, and i got bands of my desired gene, but the bands had some smears as usual due to primers and other impurities.
I stored my pcr products, before purification for 1 month at +4 degrees. Can this be the reason for the low concentration(17ng/ul) i got after purifying? I plan to sequence with both forward and backward ITS primers, hence i would need at least 40ng/ul. Is there a way i can increase this concentration.
I also used 50ul elution during cleaning, Is it possible to get a higher concentration if i decrease the elution buffer to say about 25ul?
Possibly you could make some sense out of these samples, but I'd strongly recommend that you redo the PCR. Long experience tells me (and I suspect others on this list) that it does not pay to continue experiments after questionable results. You want to do a set of reactions, verify that they worked well, and then use those as the basis for another set of experiments. If your first set are questionable, then you will never know if a later failure is due to what you are doing now is wrong, or if the previous results were wrong. Learn from our experience.
100 ng of DNA is completely common amount in PCR reaction, IF it is a a human gDNA.
(actually 100 ng of human gDNA sized 3 Gbp is around 105 copies, yet 1 ng of 1000 bp plasmid (with one copy of a gene) is almost 109 copies, so way too much )
17 ng/ul can be perfectly enough for sequencing, depending on the type and the condition of the sequencer and amplicon length (we need 7-8 ng of template for one cycle sequencing reaction of 300 bp product), BUT it's not very much for precious concentration measurement. Definitely don't use concentrations less than 10, because you can then use random number generator instead.
So, find out how much they need for sequencing.
The storagge in +4 for a month may be a problem (so far I would say the biggest problems of those mentioned). I store it there overnight, but not a month. The amplicons start to degrade from the ends (even without proofreading polymerase that actively nibbles), you may have problem with sequencing primer binding, and nonspecifities. You may get the sequence, but it would come out surely nicer, if you froze your samples at -20 or purified right away (and then froze the samples).
I personaly isolate from gel for sequencing, It's true you get lower concentration (even around 17ng/ul which is completely sufficient), but you get a clean band without remnants of template and you use it all, because you need to run a gel check anyway so why not check and isolate at the same time, the specifity is a bonus.
Increasing the number of cycles is generaly not a good idea for sequencing, more cycles are more chances of error.
For sequencing of PCR product, TA Cloning is the best solution.
As long as I know, sequencing machines are not able to read 50 bp of two tail of linear Oligo.
Why would you need to clone every single PCR product? Since I'm not getting what you wanted to say about the sequencer limitations, I would just remark that they can't read the 20-40 bp on the begining of the amplicon(depending on the type of BigDye kit version in case of ABI sequencers and purification quality of a cycle sequencing product) and the maximum read length varies with the capillary length and polymer type, however you can get reads around 450 bp with no problem and up to 900 bp.
You just design primers sufficiently far away from the sequence you need to read and have the same in mind for overlaps. But since most of the exons are not bigger than 300-400 bp you have no problems with that anyway when sequencing DNA.
I can't image what advantages would cloning/sequencing had over simple purification and direct sequencing of a a strong pretty product. It's more expensive, It's way more time consuming, and unless you sequence several clones for each amplicon (which is again more time comsuming and expensive), it's actually more error prone, since every mistake polymerase makes is 100% of a signal.
If you do PCR with a standard hotstart Taq polymerase, you will get the right sequence in 99,5 % because errors get lost in the background. It won't be usable for cloning at all, you'll need to use proofreading polymerase (and even that makes mistakes) which is again.. expensive, less processive... I can't find any advantage, really.