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CTAB buffer

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CTAB buffer

Postby sara » Fri Apr 29, 2005 7:35 am

hi there,

i have questions:

1. how long time that i can use the CTAB buffer? i mean that im prepared the 2X CTAB buffer from 4 month and kept in oven to further extraction so i need to check the pH before use or what?

2. what is meaning of 0.8% agarose or 1.5 % agarose ? that mean 0.8g agarose in 100 ml TAE buffer and 1.5 g agarose in 100 ml TAE buffer?

3. there is some problem if i use frozen plants that i keep in frezer for long time like 7 or 8 months? the extraction will be differ than fresh samples?

regards
sara
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Postby DevGrp » Fri Apr 29, 2005 1:04 pm

Yes a 0.8% gel is 0.8g agarose / 100ml buffer. You would normally use a 0.8% gel for seperating larger fragments of DNA (like genomic DNA) and a 1.5% gel for seerating smaller fragments (PCR products).

We regularly freeze plant material prior to extraction with no lost of yield / quality, again depends on the plant and the extraction procedure. How do you grind your material?

Liquid nitrogen / motar pestle ?
Bead mill?
?
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Postby sara » Fri Apr 29, 2005 1:42 pm

using 0.25g of leave tissue grind in mortar and pestle with liqiud nitrogen.

what is the meaning of bead mill is a mixer like coffee mixer?
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Postby DevGrp » Fri Apr 29, 2005 2:07 pm

The bigger bead mills used to look like coffee grinders, the one we are looking at is from Retsch http://www.retsch.de/english/db/overview.php?AKZ=E61 and is sold by Qiagen for their DNAeasy / Biosprint plant DNA extraction kits
http://www1.qiagen.com/Products/Genomic ... mples.aspx

If you plan to extract your DNA using liquid nitrogen then I would expect that freezing them first wouldn't be a problem, by choice I'd use a -80C freezer if avialable, but I don't know if this is strickly nessesary if you only have a -20C freezer.
Good luck
What are you planning to do with the DNA?
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Postby sara » Sun May 01, 2005 6:01 am

hi DevGrp

my project is to see which methods are suitable to our plant for DNA extraction by tring with many protocols then for each protocol do the restriction enzyme to see my DNA is restricted and PCR also to see if it amplified that's all.

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