Login

Join for Free!
118252 members


Protein contamination in bottle gourd DNA extraction

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Protein contamination in bottle gourd DNA extraction

Postby logan » Thu Oct 08, 2009 8:02 pm

Hey everyone,
I've been using a CTAB extraction protocol to isolate DNA suitable for PCR from single caryophillid seeds for a while now, and I recently tried the same with bottle gourd (Lagenaria siceraria, Cucurbitaceae). With the Caryophillids, I incubate the single seed in CTAB buffer for one hour, grind with pellet pestle, and proceed as normal, no liquid nitrogen is used. With bottle gourds, I've been excising a small amount of embryo tissue and attempting the same procedure, but I'm ending up with very low 260/280 values. I added proteinase K along with CTAB buffer in the last attempt, but no luck. Any suggestions? Thanks very much.

Logan
logan
Garter
Garter
 
Posts: 2
Joined: Thu Oct 08, 2009 7:53 pm

Postby JackBean » Fri Oct 09, 2009 7:13 am

I guess you are not using only the CTAB buffer, are you? So, writte you consequent steps, there must be the answer...
(the step, where you precipitate proteins and remove them or where you precipitate DNA)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby logan » Fri Oct 09, 2009 11:00 am

1) Incubate at 55C in 500 uL CTAB buffer including 0.02g PVP and 2.5 uL mercaptoethanol.
2) Grind tissue directly in buffer, add chloroform, centrifuge, recover upper phase.
3) Incubate on ice with ammonium acetate and isopropanol. spin down and pour away supernatant.
4) Wash pellet twice with ethanol and resuspend in TE buffer.

I read it's possible after DNA precipitation to add distilled water and re-precipitate proteins in 5 M ammonium acetate, so I'll give that a try. Apparently this taxon has caused other people similar problems, but I'm having issues running down exact fixes just yet.
logan
Garter
Garter
 
Posts: 2
Joined: Thu Oct 08, 2009 7:53 pm



Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests