FW: no restriction with EcoRI

Deanne Bell dbell at fresno.ars.usda.gov
Tue Aug 30 17:31:44 EST 2005

Hi Lorena

What type of DNA extraction protocol did you finally settle upon?  With AFLPs, it is very important to start out with good quality, high molecular weight, genomic DNA.

As I recall (AIR) EcoRI is the "rare cutter" and Mse I is the "frequent cutter".  So you should see lots of bands or a smear in the Mse digested DNA and very little change in your Eco digest.  What kind and %age gel are you running the digest out on?  You might try increasing the 5age for increase resolution.

Some ideas:
1) you might need to change the enzyme buffer or temperature you are using when you digest with the EcoRI

2) you might try an EcoRI isoschizomer 

3) if you find that EcoRI is not going to work, I am afraid that you will need to go back to the original 1995 Vos paper, replace EcoRI with a different "rare cutting" enzyme and have your own primers made instead of using the kit from invitrogen.

hope these ideas may help in soem way
Deanne Bell
USDA Agricultural Research Service
Crop Diseases, Pests & Genetics
9611 South Riverbend Avenue
Parlier, CA  93648
voice  (559) 596-2806
fax      (559) 596-2897
dbell at fresno.ars.usda.gov

>-----Original Message-----
>From: methods-bounces at oat.bio.indiana.edu 
>[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of 
>Lorena Quintero Barrera
>Sent: Tuesday, August 30, 2005 1:47 PM
>To: methods at magpie.bio.indiana.edu
>Subject: no restriction with EcoRI
>We are working with an amazonian plant, we wanto do AFLPs, we 
>isolate DNA and we digested the genomic DNA with HindIII and 
>MseI, but when digest with EcoRI we dont have any result, the 
>DNA is complete, there isn't restriction evidence. The EcoRI 
>enzyme hasn´t expired because the positive control (lambda 
>DNA) digest totally, we obtain the bands expected.
>So, we want to use the Invitrogen AFLP kit, it works with 
>EcoRI and MseI and if the EcoRi doesn´t work, we can't use it.
>We don't know if there are any inhibitors for the EcoRI enzyme 
>in our genomic DNA? if it so, What can we do?
>Please really need help any suggestion is welcome!!  
>Methods mailing list
>Methods at net.bio.net

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