plant DNA extraction protocol - troubleshooting (Sep/20/2006 )
hi
while isolating DNA from plant samples using the CTAB method (sandal leaves) i can visualise the DNA in the agarose gel,but its not running out of the well..i had incorporated PVPP as well as proteinase K during extraction..can any body tell me the reason for this..and also my DNA samples have lots of contaminants..can anyone help me out with a better protocol pls..
anupama
I can indicate you these papers:
Isolation of plant DNA from fresh tissue
JJ Doyle, JL Doyle - Focus, 1990
A rapid DNA isolation procedure for small quantities of fresh leaf tissue
JJ Doyle, JL Doyle - Phytochemical Bulletin, 1987
There you can find the method I use to extract DNA from Vitis leaf samples.
Hope this helps you!
Good luck!
gel not coming out of the well, basically it means that there is
1- no DNA in sample.
2- DNA is present in insoluble form. (Ie when DNA was percipitated, it had so much contamination that it didn't resuspend back into solution)
3- the DNA isn't coming out because it is very very contaminated with proteins and/or polysaccharides and/or phenolic compounds.
Might I ask, are you using the basic CTAB extraction method? You should consider combining CTAB with other purification methods. Such as proteinase K, guanidine thiocyanate based extraction, binding column extraction.
Also some variation to the CTAB protocol can be implimented like addition of PVP (Polyvinylpolypyrrolidone), followed later by emulsification with dichloromethane.
There are many protocol variations on the net. You should give them a look see.
Example
http://www.pierroton.inra.fr/genetics/labo/protocol.html
Isolation of plant DNA from fresh tissue
JJ Doyle, JL Doyle - Focus, 1990
A rapid DNA isolation procedure for small quantities of fresh leaf tissue
JJ Doyle, JL Doyle - Phytochemical Bulletin, 1987
There you can find the method I use to extract DNA from Vitis leaf samples.
Hope this helps you!
Good luck!
hi
thanx a lot for the suggestion..wil go thru the papers and hope wil get good DNA soon
anupama
1- no DNA in sample.
2- DNA is present in insoluble form. (Ie when DNA was percipitated, it had so much contamination that it didn't resuspend back into solution)
3- the DNA isn't coming out because it is very very contaminated with proteins and/or polysaccharides and/or phenolic compounds.
Might I ask, are you using the basic CTAB extraction method? You should consider combining CTAB with other purification methods. Such as proteinase K, guanidine thiocyanate based extraction, binding column extraction.
Also some variation to the CTAB protocol can be implimented like addition of PVP (Polyvinylpolypyrrolidone), followed later by emulsification with dichloromethane.
There are many protocol variations on the net. You should give them a look see.
Example
http://www.pierroton.inra.fr/genetics/labo/protocol.html
hi
among the chioces u had quoted,wat i feel is DNA is not running out of the well is mostly due to the contaminants present,may be proteins..i can find a clear band of DNA but infact inside well only..i have incorporated proteinase K as well as PVPP during the extraction,but still not enought to solve the problem..will phenol treatment help? n if so in which stage of extraction..pls lemme know
anupama
if the contamination problem is proteins then yes, phenol would help. I would do the phenol/chloroform extraction after all the other cleaning method have been used. The phenol/chloroform extration would be the last step.
If the problem is largely polysaccharides, phenol won't do much.
If the problem is largely polysaccharides, phenol won't do much.
hey
thanx for the reply..but shall i ask u somethin..shd phenol-chloroform be added as the last step,i mean to ask after DNA has been diluted in TE buffer as the final step..if so at wat concentration,can u make it a bit more clear..n also,does addition of RNase and Proteinase K at the final step more helpful than doing it in btw the steps..i usually add dem before incubating dem in waterbath..can u suggest somethin??
anupama