Hi Isar, These are two important chemicals in DNA Isolation for for removing the phenolics from the samples you are going to Isolate....
1.PVP (polyvinylpyrrolidone) is added to remove phenolic compounds from plant DNA extracts.
(PVP forms hydrogen bonds with the phenolic compounds)...
2.b-Mercaptoethanol is often included in extraction buffers designed for plant DNA extraction, because it is a strong reducing agent which can remove tannins and other polyphenols often present in the crude plant extract.
3. Let me know from which sample you are going to Isolate....
good luck.....
Hi Isar, These are two important chemicals in DNA Isolation for for removing the phenolics from the samples you are going to Isolate....
1.PVP (polyvinylpyrrolidone) is added to remove phenolic compounds from plant DNA extracts.
(PVP forms hydrogen bonds with the phenolic compounds)...
2.b-Mercaptoethanol is often included in extraction buffers designed for plant DNA extraction, because it is a strong reducing agent which can remove tannins and other polyphenols often present in the crude plant extract.
3. Let me know from which sample you are going to Isolate....
good luck.....
Hi Israr, I'm agree with Anand..I've using CTAB method with addition of PVP and I've got GOOD RESULT!! My plant DNA solution very clear, I mean after I add TE buffer on the last procedure.. if u use CTAB+PVP buffer, you should make sure that your procedure have higher salt, you can use 5M, 2M and for buffer 3.5M of NaCl. GOOD LUCK! Israr
hi israr....mercaptoethanol is basically a reducing agent that breaks the disulphide bonds in protein when running a reducing SDS PAGE electrophoresis......so if you have a protein with more than 1 subunit liked by disulphide bonds this agent can be used to find out the no. of subunits in a protein.
Hi everybody! I have problem to dissolve PVP in CTAB buffer. When I added PVP it became in a colloid solution, is it normal? Thanks!!
Hi Veronica, how much PVP do you have made? 2%? and have you solved those PVP first before mix with CTAB buffer? Because in my experience, before added PVP to CTAB, the PVP was solved by distilled water.
Erta thanks for you answer! The problem is that I cannot disolve it even in water! would be anything wrong with my PVP stock?
hmm,I don't get any idea Veronica. I guess it may caused of your PVP stock, it was expired. Just check the expired date.
@ Veronica
You are right When we add PVP it became in a colloid solution, is it normal normal only don't worry about it. and it doesn't affect your buffer.
To over come this you can just incubate your CTAB+ PVP for 15 mins at 65 C and shake/vertex, it will be dissolved completely...
good luck
Is PVP able to reduce polysaccharides as well? If not, what should I do if I want to remove polysaccharides during DNA isolation. Thanks.
Does anybody use CTAB protocol for DNA extraction from fungal mycelium?
Hello colleagues, I see that this conversation is a little bit old but maybe somebody still can answer my question about CTAB extraction protocol. I am trying to extract DNA with CTAB from fungal fresh-frozen mycelium and ran into the problem that my DNA doesn't look clean on gel when I am trying to quantify it (picture is attached, last three bands are my standards - 50,100 and 200). I noticed, that my DNA pellets are quite gelatinous even after washing with ice cold 70% ethanol (it is even hard to make a pellet from DNA after isopropanol step, in some tubes after 10 min centrifuge gelatinous aggregation stays somewhere in the liquid). And the liquid after resuspension by water is viscose (resuspension is also quite hard). So I was thinking maybe I am missing something (never used CTAB extraction before).
I started with modified protocol that my colleague suggested and this protocol doesn't have PVP and BME.
Here are all ingredients I have in my extraction:
Extraction Buffer A (EBA):
2% (w/v) hexadecyltrimethylammonium bromide (CTAB)
Tris (pH 8.0) (from 1M stock)
EDTA (from 0.5M stock)
NaCl
ascorbic acid
Extraction Buffer B (EBB):
Tris-HCl (pH 8.0) (from 1M stock)
EDTA (from 0.5M stock)
NaCl
Other Required Reagents:
20% (w/v) sodium dodecyl sulfate (SDS) (weigh in fume or with respiratory mask)
5M potassium acetate (autoclave before use, store at 4۫C)
70% ethanol
Isopropanol
On my second attempt I also added step with phenol-isoamyl alcohol to try to make it cleaner, but got the same gelatinous pellets as before.
Will appreciate any suggestions
Thank you very much
Olga
Hello,
we are using CTAB-buffer with 2% PVP for Isolation of DNA from fungal mycelium as well as from fruiting bodies with good results. This also works with the Promega Maxwell System for automated clean up.
Kind regards,
Hans-Volker
Thank you @Anad A Khot. just what I needed. A little heat and the PVP CTAB solution is now completely dissolved.
@Veronica De Pino dear De Pino, i used it in CTAB buffer and it solved easy, but maybe i used a bit warm solution of CTAB, because the amount of using it is small, so no prob for solving it, if you give some hours to it, it solved. but when you make solution from PVP, you must use it between 2 or 3 days, so better you add its powder directly to each vial for Dna Extraction, i used a 100 microliter sampleller tip and just take some powder by top of Pipette Tip and add it into each tube 1.5 cc. Please see this method that i found and used it for my samples, it was very good method for extraction of phenolic samples, i used it for Loquat ...
@Erta Puri Rosidiani yes dear friend, you true, i remember at first when i used that expired one, it was yellow a bit and result was not good, i saw not clear solution after adding this, when i understood and bought a new one, it was really white and i could take good result.
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