I am isolating genomic dna from palmyra palm (Borasus flabileffer) older leaves using CTAB method. But when I resuspend the DNA pellets in the buffer it shows a viscous jelly like appearance and during loading it into gel it won't get settled down in gel. Finally in result it shows uneven fire like band.
I have tried trick of increasing nacl concentration up to 1.5 to 2 M during precipitation of dna using ethanol but still problem persist.
Hi, Vishal, there are several way to remove polyscharides. You can find information in these papers:
http://nopr.niscair.res.in/bitstream/123456789/13514/1/IJBT%2011%281%29%2067-71.pdf
http://www.ncbi.nlm.nih.gov/pubmed/7980924
Hi, yes there are several ways to minimize contaminants from a genomic DNA extraction from plant tissues, ranging from using binding columns and silica matrices to kits, but you did the right thing in trying to increase the NaCl concentration, you could even increase it to 2.5M and make sure your phenol:chloroform is freshly made. Also you could try re-precipitate the DNA a few times including washes which may decrease the amount contaminants. Good luck :)
Hi,
try such method:
Quick freeze plant material with liquid nitrogen
Transfer 100 mg to an Eppendorf tube. Break up into a fine powder using a tooth pick or metal spatula
Add 500 мкл of 50 mM EDTA; 0,2 % SDS. Vortex or mix tooth pick
Incubate at 68 oC for 10 minutes. Vortex briefly
Spin 5 minutes in a microfuge 16 000
Transfer supernatant to a new tube and add 30 мкл 8M KAc. Place on ice for 5 minutes
Spin 5 min
Transfer supernatant to a new tube and add 600 мкл isopropanol. Mix well and spin 5 min to pellet DNA
Invert the tube to dry and resuspend in 200 мкл H2O
Add 10 мкл 10M LiCl and 500 мкл 95% EtOH. Mix and spin 5 min
Discard supernatant and dry pellet
Resuspend pellet in 30 мкл H2O
First of all you have to use freshly prepared reagents...i faced same problem in case Aloe DNA isolation...you can increase the concentration of PVP and BME, which help you a lot..after addition of TE buffer you have to incubate at 50 degree at waterbath. next day just take out the upper layer which is not viscous and proceed for gel electrophoresis....good luck
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