I currently working with M. truncatula and I am trying to perform some gDNA extraction out of leaf tissue. I have been using the following extraction buffer: 200mM Tris,pH7.5, 250mM NaCl, 25mM EDTA and 0.5% SDS. My supernatant volume is mixed with isopropanol (equal volume as my supernatant) and incubated for 10 min, then centrifuged for 7 mins. Then the pellet is resuspended with 70% ice-cold ethanol and then centrifuged again for 2 mins. I let my pellet dried and then resuspended with ddH2O.
I have used this method and always, my pellet turned out orange or pink. I am trying to obtain good quality of gDNA. Any suggestions?
Try Murray and Thomson 1981 protocol. I am using this protocaol since 2003 and I have got good quality of gDNA always whether it is plant, Bacteria or fungus. The Protocol is as below
Tris HCl pH 8.0 ------------------- 100 mM
NaCl-----------------------------------1.4M
EDTA pH 8.0-------------------------20mM
CTAB------------------------------- 2%
PVP--------------------------------1%
Beta meceptaethnol-----------0.5%
Heat samples at 65 deg for 1 and Half hours with constant starring
Cool the samples and add chloroform isoamyl alchol 24:1 and agitate
spin at 10000 rpm for 15 min
collect supernatant and transfer to other tube and then add chilled isopropanol (equal vol.) and left at -20 deg for overnight
centrifuge and wash the pallet with 70% ethanol, dry and suspend in TE buffer
Hope it will yield good quality DNA
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How much elution buffer volume should i use? or you recommend? TE buffer pH=7.5, right?
I worked with Aloes and gDNA extraction is quite problematic from these species, resulting in high level of contaminations. I agree with B.A Padder, that the steps with chloroform and isoamyl will decreased the level of contamination. I am using the protocol in the paper attached
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