Standardizing DNA extraction protocol for whole blood samples!

[AlexBiohazardous]

I have been working on DNA extraction from blood for about 4 months voluntarily in the biology lab of my university(medical school in greece).

My professors there have managed to figure out a stable home-made salting-out protocol for most blood samples(there have been some ''extreme'' exceptions where a kit was used).So I sticked up with it and managed to succesufully extract DNA from about 50 samples (0.5ml fresh blood each).

Let me be more specific as to the reason I posted this.I was recently asked to standardize the protocol to produce higher yields in less time(less steps that is) and with less reagents to make it cheaper(remember, it's home-made protocol).

So, I did a research....
After 3 days,I realised that the sources I was able to access were proposing sligtly different approaches(with the exception of the Et-OH precipitation step) and I was more than confused.

I have tried to implement a new protocol with some major changes but the results were not as promising.I had enough quantity but the DNA was degraded and with abnormal ratios(260/280, 260/230).

Here are the problems I faced:

- The clock is ticking : the old protocol lasts 3 whole days because the purification of the DNA was better.Trying to shrink it back to 2 or even 1 day would mean compromisation of the quality.So,you may ask,why 3 days?There 2 elements of the process that led to this time which I describe below.

- The power of the enzyme :According to the protocol,only through an overnight digestion with Pr.K or some other enzymatic way,can the proteins denature properly.I am trying to find a way around it through a non-enzymatic denaturation(which will be cost-effective,since the enzyme costs a fortune ).I dug up some non-enzymatic protocols in my research but most were using phenol-chloroform afterwards.However,I understanded that SDS(for fats degradation) could easily denature proteins.

- Up in the sky: after the ethanol precipitation the tubes are left open overnight so the DNA can dry properly.A solution around this would be to use a hood,but I was told that I could actually lose in DNA yield,if I forced the reamaining ethanol out of the eppendorf.

- Salty stuff: The ratio 260/230(the presence of salt together with DNA) has been steadily falling from about 1,5 to 1,2 to 1 to 0,5 ,meaning something was going wrong in the removal of salt with the 70% Et-OH.Increasing the washes with the ethanol meant a lower DNA yield.Beacuse of that,the blame had to be given to the stock of the 6M NaCl.I personally made a new stock(200ml) but the problems continued.

After leaving the lab(for summer) without good results,I decided to search even more.Then I found this forum.I hope you can help me build a non-enzymatic low-cost and low-time protocol.Any advice or tip is welcome.

Thanks in advance
Alex

ps: if you have any question on the rest of the protocol I use,just ask.

[luisillo]

I have a protocol but I have it in spanish. I can translate it to english but it would take me a couple of days. The protocol involves two detergents (DTAB and CTAB) and chloroform, as well as ethanol precipitation. Reply me if you are interested.

[AlexBiohazardous]

I am aiming for a salting-out process(not involving chloroform) but I would like to see that protocol!!I'll be waiting for your answer.

[luisillo]

Here's the protocol I told you. Sorry for the BIG delay, job issues you know. Anyway, I hope you can put it to good use xD.

1. Extract blood in EDTA tubes (purple top).
2. Add 400 ul of DTAB buffer (8% DTAB*, 100 mM Tris pH 8, 1.2 M NaCl, 50 mM EDTA) to 300 u of blood and incubate at 65ºC for 5 min.
3. Add 500 ul of chloroform and quickly close the tube and vigorously shake the mix for at least 5 min. This is a critical step, if you don't do it fine you will have blood clots in your mix which will decrease the quantity (and probably the quality) of your DNA.
4. Centrifuge the tubes at 13,000 rpm for 5 min.
5. Transfer the aqueous phase to a new tube and add 100 ul of CTAB buffer (5% CTAB**, 400 mM NaCl) and 900 ul of sterile deionized water (or nuclease free water). Mix gently and incubate at -20ºC for 10 min.
6. Centrifuge at 10,000 rpm for 5 min and discard the supernatant.
7. Resuspend the pellet in 200 ul of 1.2 NaCl and 300 ul of cold 100% ethanol. Mix gently (you might be able to see the nucleic acid precipitate).
8. Centrifuge at 10,000 rpm for 5 min and discard the supernatant.
9. Wash the pellet with 700 ul of 70% ethanol.
10. Let the etOH evaporate and resuspend the pellet in either nuclease free water or TE buffer.

* Dodecyltrimethylammonium bromide
** Hexadecyltrimethylammonium bromide

[AlexBiohazardous]

Originally Posted by luisillo

Here's the protocol I told you. Sorry for the BIG delay, job issues you know. Anyway, I hope you can put it to good use xD.

1. Extract blood in EDTA tubes (purple top).
2. Add 400 ul of DTAB buffer (8% DTAB*, 100 mM Tris pH 8, 1.2 M NaCl, 50 mM EDTA) to 300 u of blood and incubate at 65ºC for 5 min.
3. Add 500 ul of chloroform and quickly close the tube and vigorously shake the mix for at least 5 min. This is a critical step, if you don't do it fine you will have blood clots in your mix which will decrease the quantity (and probably the quality) of your DNA.
4. Centrifuge the tubes at 13,000 rpm for 5 min.
5. Transfer the aqueous phase to a new tube and add 100 ul of CTAB buffer (5% CTAB**, 400 mM NaCl) and 900 ul of sterile deionized water (or nuclease free water). Mix gently and incubate at -20ºC for 10 min.
6. Centrifuge at 10,000 rpm for 5 min and discard the supernatant.
7. Resuspend the pellet in 200 ul of 1.2 NaCl and 300 ul of cold 100% ethanol. Mix gently (you might be able to see the nucleic acid precipitate).
8. Centrifuge at 10,000 rpm for 5 min and discard the supernatant.
9. Wash the pellet with 700 ul of 70% ethanol.
10. Let the etOH evaporate and resuspend the pellet in either nuclease free water or TE buffer.

* Dodecyltrimethylammonium bromide
** Hexadecyltrimethylammonium bromide

Many thanks!
I have some questions about this protocol:

1.What is the starting volume of whole blood from the EDTA tubes?You have specifically mentioned the other quantities,but what about the starting one?
2.In the lab that I worked,extraction takes place in 1.5ml eppendorf tubes(0.5 ml is collected from the EDTA tube).Will this affect the protocol viability in any way?I will try to fit the quantities of the reagents to 500ul blood.If you can help out it will be much appreciated.
3.I am bit baffled by the protocol.It doesn't involve phenol but it does involve NaCl (like in salting-out protocols).Is it some sort of hybrid
4.I have quickly measured how much time is needed and I can see it's about one hour from the whole blood to the spectrophotometer which is easily comparable to kits.The question is,will I extract,over 200ng/ul, high quality DNA?

If you happen to have any more protocols for whole blood DNA extraction,I'd like to see them too.

Again,MANY thanks!!
Alex

[luisillo]

1. The starting volume is 300 ul of blood for each 1.5-2 ml eppendorf tube (I suggest you run the assay by duplicate or even triplicates, since the starting volume is low).
2. I do not recommend you to rise the volumes if your working with 1.5 ml eppendorfs since the reagents won't fit in the same tube well enough to mix properly. However, if volume isn't an issue for 500 ul of blood you'll have to use 650-700 ul of DTAB (rise the incubation time a little should also help since is more blood) and around 800 ul of chloroform. For the next steps you can either increase or not the volumes by a 50 percent (i.e. 100 ul to 150 ul)
3. I suppose so. My former boss taught me the protocol, but I didn't checked the original paper. I'll give you the reference if you're interested. I warn you: the protocol I put here is a modification of the original. Gustincich S, Carmici P, Del Sal G. A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques 1991;11:74
4. If done well you will get 50-100 ng/ul per tube (when you dilute pellet in 50-70 ul of TE or water). About the quality, the spectrophotometer told me (almost always) is good xD. It was fine to run conventional PCR, which was what I used to do with it. I think the abstract of the paper tells the protocol gives, as I mention, reasonable yields and quality considering time and money expenses.

We used to do another protocol with whole blood, but it was the standard SDS-proteinase K, phenol-chloroform 3 day protocol.

Good luck