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10 Questions you want to ask about PROTEINASE K.

  
  
  

1. QUESTION: What is Proteinase K?

ANSWER: In molecular biology Proteinase K (also protease K or endopeptidase K) is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8. The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa).
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2. QUESTION: What is the function of proteinase K in DNA extraction?

ANSWER: During the extraction of DNA (or nucleic acids in general), there is a lot of contaminating proteins present. These contaminants must be removed. Proteinase K, which is a broad spectrum serine protease, is used in many DNA extraction protocols to digest these contaminating proteins.

In addition, there may be nucleases (enzymes that degrade nucleic acids) present. The addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction.

 

3. QUESTION: What are proteinase K applications?

ANSWER: Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid. Addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. It is highly-suited to this application since the enzyme is active in the presence of chemicals that denature proteins, such as SDS and urea, chelating agents such as EDTA, sulfhydryl reagents, as well as trypsin or chymotrypsin inhibitors. Proteinase K is used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. Some examples for applications: Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5 - 1% SDS. Purification of genomic DNA from bacteria (miniprep): bacteria from a saturated liquid culture are lysed and proteins are removed by a digest with 100 μg/ml Proteinase K for 1 h at 37 °C. The enzyme's activity towards native proteins is stimulated by denaturants such as SDS. In contrast, when measured using peptide substrates, denaturants inhibit the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease.


4. QUESTION: Why is proteinase K digestion performed at 50°C?

ANSWER: Proteinase K activity is greatly increased by addition of denaturing agents like SDS or urea (Hilz et al., 2008), indicating that the denaturation of the substrates helps Proteinase K to degrade them. Increasing the temperature to 50°C will also unfold some proteins already, making it easier for the Proteinase K to degrade them. The proteinase K seems to be a pretty stable enzyme, and can still work at this temperature.

 

5. QUESTION: What are temperatures proteinase k inactivated?

ANSWER: Proteinase K is inactivated by heat, eg. Incubating at >55 °C.

 

 

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Comments

Hello everybody I just found your website, which I find very useful! I have a concern regarding proteinase K, I normally use it at 10micrograms per ml of PBS and works perfect at the moment to perform insitu hybidization in fish, however last time I prepared more and it failed since my fish did not present any staining. I did noticed that 3 days after I have started my insitu for that reason I would like to know wheter there is a method to test proteinase K works before using it. Kind regards Maria Romero
Posted @ Saturday, October 13, 2012 5:48 AM by Maria
To Maria Romero. 
If you have any albumin or some other protein, you can use the Proteinase K on the Albumin first and run a gel comparing the Proteinase K + Albumin versus a control of just Albumin and a control of just Proteinase K.  
 
If the Albumin was digested you should get plenty of bands in your gel.  
Proteinase K will also cleave benzoyl arginine-p-nitroanilide, which gives you a yellow p-nitroaniline. This can be detected at 410 nm on a spectrophotometer. This is an activity test that is sometimes done for varius enyzymes. 
 
You can compare activity of your Proteinase K over time with the spectrophotometer method to check the activity.  
 
You can compare the Proteinase K on the day you prepped(opened) it, versus the next time you use it. 
 
The typical QC method to determine units for Proteinase K is that one unit will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 μmole of tyrosine per min at pH 7.5 at 37 °C. One protocol for that can be found at http://www.worthington-biochem.com/PROK/assay.html 
 
You could also call up the manufacturer and ask them what does the COA says and find out how they determine activity. 
 
Your Proteinase K activity could have been inhibited by EDTA, DIFP, or PMSF. So you might want to check for those type of containments. 
If the pH was very low or if the temperature range was too high or too low, that could have been an inhibitory issue as well. 
 
Good Luck 
David Starr 
 
Posted @ Tuesday, November 27, 2012 12:19 PM by David Starr
hello.i have a problem in my DNA extraction, and have weak band in pcr product,now i want to use protein kinase k to remove proteins,i wanna know that`s right way? ichecked primer and other material.any suggestion??? tnx alot. 
Posted @ Monday, January 21, 2013 6:57 AM by fatima
Fatima, 
I do not understand what you mean by a weak band in pcr product. To clarify, does this mean you are doing PCR on your extraction and then taking the replicated DNA and separating it with a gel to get a band?  
It would also help to know what tissue you are extracting the DNA from and the method you are using to extract the DNA. In most cases the proteinase K is usually used on the tissues BEFORE the DNA extraction process. Frequently it will be a digestion process between 37 to 55 degrees from 1 hour to overnight or over weekend time periods. This mixture then might be heated to 95 degrees for a small period of time to inactivate the Proteianse K. This digestion process may be done with various detergents added to allow for proper digestion of any proteins. Some of those detergents may also cause interference. I have read of SDS interference being inactivated by Tween 20. As for your primers, I am sure they are fine. Your Master Mix might be an issue. How sensitive is your master mix to temperature changes? What is the contents of the master mix. Perhaps switching out dTTp with dUTP and using UNG will help with any amplicon carry over and false positives. 
Posted @ Monday, January 21, 2013 6:39 PM by David Starr
think you all, but I have a question. Would this proteinase K used as Antigen Retrival for anti-body specific binding? 
Posted @ Tuesday, June 25, 2013 3:35 AM by Dana
There are currently methods out there for use for of Proteinase K in antigen retrieval. My worry would be high concentration levels or excessive contact times affecting tissues you would not want to be affected. In any case here is a link I saw for some methods. FYI I don't work for this company, I just tend to answer questions on this thread.  
http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm 
 
I think the method 2 might be better. If it was a learning process and your tissues are valuable I would be cautions and go with lower temperatures and contact times and adjust upwards until you find a happy medium. 
Posted @ Tuesday, June 25, 2013 10:14 AM by David Starr
Hi! I'm working with fecal samples and I'm trying to get DNA from Eukaryotic parasites. I have read in some articles that they use proteinase K as a really important step during the DNA extraction. May I use Proteinase K if I'm using phenol:chloroform:IAA(25:24:1, pH7.9) as part of the protocol? when should I add the proteinase K?
Posted @ Friday, July 05, 2013 4:04 PM by Melissa
Proteinase K is used in DNA extraction as it digests proteins especially nucleases that may degrade DNA/RNA. Activity of protienase K is enhanced in presence of SDS. Proteinase K can be used with phenol:chloroform:IAA protocol and it is generally added after the cell lysis step. The cell lysate to which proteinase K has been added is then incubated for 2-3 hours. The time and temperature for Proteinase K activity might have to be optimized. -Pratima Doshi, Guest Blogger
Posted @ Monday, July 22, 2013 9:57 AM by ASHLEY LEE
I'm using Phusion polymerase to generate inserts with restriction enzyme sites for standard cloning and having difficulty getting the inserts in. I'm wondering if the polymerase may still be active in the digest (I run the pcr over a spin column before digesting) and filling in the ends or chewing back the ends after digestion. Has anyone used PK to remove Phusion prior to digestion with Restriction Enzymes and is there a protocol available? 
Thanks!
Posted @ Tuesday, August 06, 2013 10:03 AM by Dianne Duncan
However, optimal concentration for resistant clones selection in mammalian cells depends on the cell line used as well as on the plasmid carrying the resistance gene, therefore antibiotic titration should be done to find the best condition for every experimental system.
Posted @ Friday, September 20, 2013 8:45 AM by essay writing services
What is the difference between using β-mercaptoethanol and proteinase K when extracting RNA and DNA?
Posted @ Thursday, October 03, 2013 3:33 PM by nazly
I am doing DNA extraction with chelex with eel tissue.The protocol is to add Proteinase K right after adding the chelex. However, today I waited for more than 10 minutes after the chelex before I added Proteinase K. Is it going to affect the purity of the DNA?
Posted @ Thursday, January 23, 2014 7:22 AM by Ling
Ling, 
Chelex is used to protect the DNA from the heating process that is used with Proteinase K. It does with a binging of Mg2+ ions during extraction.  
It will not damage your DNA. 
10 minutes of time should not affect your DNA purity at all.  
This is assuming everything was done at room temperature. If you were extracting for RNA, a time crunch might be an issue. 
Posted @ Thursday, January 23, 2014 8:38 AM by David Starr
"Binding" of MG2+ not "binging".
Posted @ Thursday, January 23, 2014 8:39 AM by David Starr
Oh thanks for the prompt reply. Hopefully it is not gonna give a big difference like you said. Are you in the line of molecular biology?
Posted @ Thursday, January 23, 2014 9:07 AM by Ling
I have over 10 years of Biotech experience and over 3 years of extraction experience.
Posted @ Thursday, January 23, 2014 8:34 PM by David Starr
I have further questions about my labwork, can I please contact you on your email?
Posted @ Tuesday, February 18, 2014 8:13 AM by Ling
I would prefer if you asked the questions here. Peer review is a very powerful tool. If you promise not to be embarrassed to ask a question. I promise to not be embarrassed to say when I don't know the answer.  
If this is a proprietary issue, I might not be the correct person to ask.
Posted @ Tuesday, February 18, 2014 8:28 AM by David Starr
We are supposed to isolate DNA from full blood samples that have been stored for several years. Despite anticoagulants most samples are clotted. How could we prepare those samples for automated pipetting (>10.000 samples). Would proteinase K work? Other enzymes?
Posted @ Friday, February 21, 2014 8:03 AM by Vinzenz Lange
I am not sure how you are doing the isolation. Some methods vary. Although Proteinase K would definently be helpful, it could be done during or after the Lysis and wash steps. Just remember Proteinase K usually works better in above room temperature applications. There are various lysis buffers as well. I am not sure which you are using.  
In most cases you would take the blood samples out of the cold and if frozen leave them out over night to thaw. This will also help the blood cells to lyse. 
 
Normally if blood has not been stored for a long period of time or frozen, pipet agitation can be used, but in this case the clotting can be broken up with vortexing. Considering the storage time, I would think that would be best. Sorry, you are going to have to do manual labor to get the best results.  
Make sure the tubes holding the blood are well sealed and have no cracks.  
Inside a a biosaftey cabinet or hood place a vortexer and individually place the tubes on the vortexer one or two at a time and try to break up the clots. 
You will then want to do a spin down 5 to 10 minutes at around a few thousand rpms. 
You would then discard the supernatant. 
At this point you might do a lysis buffer step. You could add a certain amount of lysis buffer with protenase K and cap the tube and vortex like crazy to break up the pellet and clots. An incubation period can occur at this step. 
Another spin down would occur.  
The supernatant would get discarded and then you can do your wash and elution steps. 
Also be sure to make sure your controls go through the same process. (Even if they don't need it.) 
If you look up ChargeSwitch® gDNA Purification Kits from Life Technologies you can see a protocol where they use Proteinase K with the Lysis buffer and it is done at room temperature. You can look around the net for other blood with proteinase K extraction or isolation methods. I hope this helps. 
 
I hope this helps.
Posted @ Friday, February 21, 2014 12:31 PM by David Starr
Thanks David for your detailed thoughts!  
I am a little surprised that you suggest to discard the supernatant after the first spin and even after proteinase K treatment. I would have thought that after extended storage times many cells would have been lysed and a large fraction of the DNA would end up in solution/supernatant. What makes you confident that even after centrifugation and lysis the/most DNA would end up in the pellet?
Posted @ Monday, February 24, 2014 5:12 AM by Vinzenz Lange
Vinzenz asked. "What makes you confident that even after centrifugation and lysis the/most DNA would end up in the pellet?" 
 
All I can say is experience and the testing of the Optical Density (OD) of the DNA versus a blank of the eluent solution.  
Also various types of Electrophoresis to determine quality. 
The Agilent Bio analyzer, etc. 
There are various types of extraction/isolation methods out there, I am just trying to help you figure out how to deal with the blood clots and if and when to use the Proteinase K.
Posted @ Monday, February 24, 2014 8:21 AM by David Starr
Thanks! 
Nothing beats experience! We will give it a try...
Posted @ Monday, February 24, 2014 10:34 AM by Vinzenz Lange
I wish I had a better option for you with the 10,000 plus samples you have to deal with, but most clots are going to need some type of serious agitation to be broken up. Heat sources can add energy to the process and reduce the agitation needed, but at the same time that might affect the DNA quality.  
 
Simple up and down automated pipeting has not worked well for me in the past and the tips could easily get clogged. I also have no idea what type of extraction/isolation method you are going to use on the eluent and the time constraints.  
Good Luck
Posted @ Monday, February 24, 2014 4:48 PM by David Starr
I was just wondering if anyone could help me, hopefully I will get it in time as I left this question last minute. I know with the Qiagen DNeasy kit you put buffer ATL in with your tissue samples and then add proteinase K and then incubate at 56oC. I have however added buffer ATL already to my tissues then realised I don't have enough room in my waterbath for all my samples. Is it ok to leave the tissues in buffer ATL for a day or 2 when I can fit them all in etc? Or should I add proteinase K to them, vortex and then just store it at room temp? OR freezer even? Any help would be much appreciated, Thanks!!!
Posted @ Monday, March 03, 2014 7:13 AM by Narelle
The Proteinase K is much more effective at the 55 degrees setting. ATL contains detergents that will help unfold the proteins allowing the Proteinase K to be more effective. The ATL should not degrade your DNA. Room temperature on the other hand could help certain organisms possibly replicate in your sample. So cold storage or freezing might be a good idea. You should be able to store the samples with the ATL in the cold and add the Proteinase K later. I would suggest -20 Freezer for long periods of time. 
I don't know what your samples are being used for, but it is good science to keep certains steps consistant with each other. This way you can keep your control samples to a minimum.
Posted @ Monday, March 03, 2014 3:05 PM by David Starr
Thanks David, Yeh the kit says at 56oC but I have heard varying temps for the incubation step. I ended up putting it in a -20oC freezer overnight. The reason why I put them all in ATL was because I had heard that you can leave it at that step and go back but then the book says after proteinase K digestion you can store it in buffer ATL at room temp for 6 months. Noone had told me about the 'after proteinase K' bit. I am going to try and borrow a massive water bath for the next samples so they can all fit, unfortunately wasn't able to last night. I normally do it consistently and in small batches but yesterdays was quite large. Thanks again for your help :)
Posted @ Monday, March 03, 2014 9:23 PM by Narelle
No problem. In the past I have usually ran the proteinase K assay at 55 degrees or 37 degrees celcius. The 37 degrees was usually for over the weekend digestion while the 55 degrees was usually for shorter digestions.  
 
I have extracted DNA out of samples that have been frozen at -20 AFTER the Pro K digestion step. 
It is fine, but it might not be good science. I have never had samples sit at room temperature with Pro K in them for an extended period of time. Keeping things at room temperature can allow other organisms to grow in your samples. 
 
The thing is you want to have a good control with defined stop steps. So you might want to avoid situations where the control does not go through the same steps as the samples.
Posted @ Tuesday, March 04, 2014 3:48 PM by David Starr
may i know if proteinase K could degrade my DNA samples as when i'm doing my extraction, i've misculculate the exact amount i should put on my samples. My supervisor said, it will not give effect on my samples but then during electrophoresis, the band doesn;t seen so clear, and then, my sv said it might be due to the numerous amount of Proteinase K..is it true that it will degrade our DNA samples?
Posted @ Wednesday, March 05, 2014 9:21 AM by shuhada
On one of my jobs in the past somebody added 10x the amount of Proteinase K than they should with no ill effect to the sample. 
I would think no. How concentrated are you talking about? 
I doubt the Proteinase K might affect the acrylamide gel you are using in your electrophoresis. 
I guess you could experiment with that by running various concentrations of Proteinase K with the marker through a gel. 
Do you have any other way to test your sample? Nano drop? Spec? Agilent Bio Analzyer? etc 
Posted @ Wednesday, March 05, 2014 9:36 AM by David Starr
Hi,  
I am trying to isolate genomic DNA from green algae called Chlamydomonas. I was given the protocol to try it put but I have a problem with low yield and RNA in my sample. The protocol has a Lydia buffer consisting of nuclease free water, Tris/HCl pH 8, NaCl, EDTA, 2% SDS, Qiagen  
Proteinase K, and PVP 1%. I am using cell pellets  
that are frozen on try ice and each cell pellet is equal to 1.5 X 10^8. One 1mL of lysing buffer is used for each pellet and pipetting 12 times is used to dissolve the pellet. The phenol:chloroform ph 7.9 is used and incubate at room temperature for 5 minutes the spin for 5 minutes. My question is if it's okay to used proteinase K in this lysing buffer. Can you suggest a better order to used proteins K or a better lysing buffer.
Posted @ Thursday, March 06, 2014 9:49 PM by Alba
I see some issues that need to be addressed. 
The first issue is RNA interference. 
The second issue is low yield. 
The third issue is the use of Proteinase K. 
The fourth issue is the buffer. 
 
About that RNA interference; Although, I think your buffer pH might be better between 8.5 to 9 pH for the Phenol:chloroform extraction. This is based on experience. I have read how pH in chloroform extraction can be adjusted to separate DNA from RNA, but I am not an expert on that. I will steal these words from Wikipedia. 
In brief, aqueous samples are mixed with equal volumes of a phenol:chloroform mixture. The proteins will partition into the organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the aqueous phase. If the mixture is acidic, DNA partitions into the organic phase while RNA remains in the aqueous phase.  
This is why I would advise a more basic pH, you might be losing some DNA. 
I would also advise including an RNAse step. RNAse is short for ribonuclease A. It will break down the RNA without affecting the DNA. I checked AG Scientifics website for it and found it priced at 0 dollars. I am going to assume by that price that they don’t have it so feel free to check out http://www.lifetechnologies.com/order/catalog/product/12091039 where I do know they do sell it. This RNAse step is usually done AFTER the break down steps and incubation but before final isolation. You might only need about 2 to 5 uL per sample for a very small time period ~ 5minutes. 
The lysing buffer is designed to denature the proteins and break up the cellular walls to allow for a release of the DNA and other material in the targeted material. Proteinase K will not harm your DNA and can be added with the lysing buffer. To increase your DNA yield you could increase your buffer/Proteinase K contact time with the target. The higher the temperature and contact time with Proteinase K, the higher the effectiveness.  
Posted @ Friday, March 07, 2014 11:28 AM by David Starr
Thanks for the feedback! So my protocol does have a RNase A incubation at 37 C for 20 minutes, step after the phenol:chloroform step.  
 
Are you advising I use a more basic buffer? Thanks!
Posted @ Friday, March 07, 2014 2:31 PM by Alba
If you have a RNase step, I am puzzled why there would be RNA interference.  
Most kits are set up with certain conditions that have most likely been optimized by the manufacture of the kit. At this point, I have to wonder if you are using a Kit. If you are using a Kit, I would think calling the manufactuers customer service is the best bet. IF not, then yes I would raise the pH to the 8.5 to 9 range. 
 
 
 
Posted @ Friday, March 07, 2014 3:56 PM by David Starr
The second portion of the protocol is a kit that comes with RNase A in it. Instead of another phenol:chloroform step, a kit is being used to removed the remaining debri after RNase A incubation step. Thank you very much for the feedback!
Posted @ Sunday, March 09, 2014 1:14 PM by Alba
Hello! I'm very happy to see that there is a place like that to help us in moments of almost despair LOL 
Well, lets go to the question! I'm having trouble to succeed a good extraction of some Enterococcus strains. I don't have any kit, and I'm trying to make a home-made procedure, using CTAB detergent. Can I use Proteinase K with it? The protocol suggests that I incubate my samples with CTAB at 60ºC. Proteinase K can handle this temperature? And overnight? Or should I try SDS?
Posted @ Monday, March 10, 2014 8:28 AM by Wal
As far as I Know Proteinase K can handle 60 degrees but 55 tends to be ideal and higher temperatures it is not as effective. 
CTAB Cetrimonium bromide is a Cationic surfactant. SDS Sodium dodecylsulfate is an anionic surfactant. Your bacteHydroxide with the SDS? Is Chloroform involved? Honestly, unless I saw the whole protocol, I would not even know where to began. Proteinase K might not be needed if using the NaOH. It doesn't hurt, but you are just breaking down bacterial strains. do you need the cells completely broken down and lysed? 
ria is a gram positive bacteria and has a single lipid bilayer. Enterococcus is also very tolerant of high sodium environments and up to 45 degrees celcius. 
Are you using Sodium Hydroxide? Are you using a chlorophyl or bead method? 
I don't like reinventing the wheel so I am just going to cut and paste from another website.  
http://www.askabiologist.org.uk/answers/viewtopic.php?id=6911 
 
SDS, an anionic surfactant (often found in many cleaning products like hair shampoo), does lyse the bacteria. In alkaline lysis plasmid DNA minipreps SDS is often used in conjunction with NaOH (e.g., 0.2N) following a lysozyme enzyme digestion, and then you would not typically have a proteinase K step. PK is more often used with genomic DNA isolation (without alkaline lysis) or even after a restriction enzyme digest. So proteinase K will inactivate nucleases (e.g., DNases/RNases) particularly in the presence of SDS, but it a broad-spectrum protease that also digests proteins to ensure complete lysis of the cells.  
 
CTAB (Cetyltrimethylammonium bromide) is a cationic surfactant that further solubilizes the cells. In general DNA is ‘happy’ in NaCl - the Na+ ions do neutralize the negatively charged phosphates on DNA and facilitate DNA molecules coming together (the molecules are less hydrophilic). A high concentration of NaCl is required otherwise CTAB-nucleic acid precipitates can form - what you are trying to do here is remove all the junk (bacterial cell wall debris, denatured proteins, polysaccharides) complexed with CTAB and leave the bacterial DNA in solution 
 
Posted @ Monday, March 10, 2014 7:27 PM by David Starr
Oops I did some bad editing on the above post. 
I was trying to ask if you would use NaOH with the SDS. 
Anyway good luck.
Posted @ Monday, March 10, 2014 7:29 PM by David Starr
There is a powercut of 20minutes in the University today and I had a PCR reaction running. The reaction resumed after 20minutes, I am wondering if the PCR going to work?
Posted @ Friday, March 14, 2014 9:10 AM by Ling
I am sure it worked to a point, but that does not mean it worked effectively to be usable for any sort of provable scientific data.  
PCR has various heating and cooling steps. IF the power went out during one of the stages where high heat was needed, your cycle will have issues. When the temperature dropped from the extension/elongation phase it would have most likely dropped into an annealing phase and than dropped to a colder level. 
Even if everything was perfect, I would never use that data. 
Posted @ Friday, March 14, 2014 10:44 AM by David Starr
Thank you. The band does show but to be sure I wont use this for sequencing.
Posted @ Friday, March 14, 2014 4:53 PM by Ling
Hello, and thanks for the information that is posted. I would like to find out what protein should be used to lyse the bacterial cell in DNA isolation? would it be Proteinase K or lyticase? and secondly please explain to me what is 16SrDNA and how it works? 
 
Thanks Much. Donie
Posted @ Saturday, March 22, 2014 1:50 AM by Candy Mars
Donie, 
Lyticase is normally used for the isolation of DNA from yeast and fungus, so given the choice of using Proteinase K or Lyticase on bacteria I would use the Proteinase K. 
I am not sure what you are asking about 16SrDNA. The S in 16 S stands for Svedburg unit and is the number is based on sedimentation rates of a particle, with the larger numbers settling faster. rDNA is a DNA sequence that codes for ribosomal RNA. Ribsomes are organelles and are usually the primary site for biological protein synthesis in cells. Since species and strains of bacteria usually need very specific proteins for their functioning, we can usually find strands of DNA very specific to a species in ribosomal DNA. 
18s rRNA is found in eukaryotic DNA and its counterpart 16s rRNA is found in Prokaryotes and mitochondria. So a test for 16s rDNA using specific primers and probes, DNA isolation, amplification, sequencing, and electrophoresis can create a specific test to identify Prokaryotic bacteria strains and species against a known data base. 
If you would like to check out a good protocol and test for 16s rDNA please check out 
http://www.lifetechnologies.com/order/catalog/product/4346479?ICID=search-product 
Go down to manuals and products and feel free to read the pdf files for the manual and protocol to get a better understand of the process and procedures involved. 
Posted @ Monday, March 24, 2014 2:39 PM by David Starr
Could somebody help me with the best conditiond for Proteinase K to autodigest in the process of digesting other proteins in the solution. Basic aim is the have a final solution with least protein remaining. Thanks.
Posted @ Thursday, March 27, 2014 1:18 PM by Nell
I would say 3 to 8 hours in a proper lysing solution at 55C in a thermal shaker
Posted @ Friday, March 28, 2014 12:27 AM by David Starr
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