Scribd
Upload
46 views2 pages

Plant DNA Isolation Protocol Guide

1) Grind leaf samples into a fine powder and add them to prewarmed CTAB buffer, incubating for 1 hour to isolate DNA. 2) Centrifuge samples and collect supernatant, adding chloroform:isoamyl alcohol to extract DNA. Precipitate DNA with iso propanol and sodium acetate overnight. 3) Wash DNA pellets with ethanol, resuspend in TE buffer, and treat with RNAseA to purify DNA for downstream applications such as PCR or sequencing.

Uploaded by

GiselleLorenaFlorezLopez
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
46 views2 pages

Plant DNA Isolation Protocol Guide

1) Grind leaf samples into a fine powder and add them to prewarmed CTAB buffer, incubating for 1 hour to isolate DNA. 2) Centrifuge samples and collect supernatant, adding chloroform:isoamyl alcohol to extract DNA. Precipitate DNA with iso propanol and sodium acetate overnight. 3) Wash DNA pellets with ethanol, resuspend in TE buffer, and treat with RNAseA to purify DNA for downstream applications such as PCR or sequencing.

Uploaded by

GiselleLorenaFlorezLopez
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

1) DNA ISOLATION FROM PLANT LEAVES SAMPLES

BY MAXI PREP

Take minimum 1g of leaf sample and grind it with the help of mortar and pestle
make them a fine powder and add 15ml of prewarmed working CTAB buffer at
650C.
Incubate the tubes at 650C for one hour.
Place the tubes an ice for 10 min and centrifuge 10000rpm 10 min at 40c.
Collect the supernatant and add equal volume of chloroform:isoamyl alcohol
(24:1), mix and place on ice for 5 min.
Centrifuge at 10000rpm 10min at 40c.
Collect the supernatant and precipitate with excess of iso propanol and 3M
sodium acetate (100µl) overnight.
Centrifuge at 12000rpm 15min at 40c.
Retain the pellet and add 5ml 70% ethanol and incubate on ice for 5min.
Centrifuge at 12000rpm 10min at 40c.
Dry the pellet at room temperature and add 1ml of 0.1x TE buffer, mix gently
and aliquots in 2tube.
Add 10µlRNAseA (10mg/ml) and incubate at 370c for 1hour and at 650c for
15min.
Place on ice for 5 min and add equal amount of phenol:choloform:isoamyl
alcohol ([Link]) mix gently place on ice for 5 min.
Centrifuge at 10000rpm 10min at 40c.
Collect the supernatant and add equal amount of choloform:isoamyl alcohol
(24:1), mix gently and place in ice for 5min.
Centrifuge at 10000rpm 10min at 40c.
Collect the supernatant and precipitate with ethanol and 3M sodium acetate
(50µl) overnight.
Centrifuge at 13000rpm 15min at 40c.
Retain the pellet and add 1ml 70% ethanol and incubate on ice for 5min.
Centrifuge at 13000rpm 10min at 40c.
Dry the pellet at room temperature and add 0.1x TE buffer, mix gently and
place on ice for longer time to dissolve pellet.

Note: It’s better to use cut tips to avoid degradation

You might also like