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hygromycin resistance

Zhiyong Wang zywang24 at stanford.edu
Tue Jun 29 19:38:12 EST 2004


We use 20 or 25 mg/l hygromycin. Another good news I forgot mention: 
you can screen much more seeds per plate in the dark than in light. 
We usually screen three to four thousand seeds on each 150 mm plate.

Zhiyong

>Your arab-gen net posting with your hygromycin protocol was very 
>interesting.  I think I'll give it a try.  What concentration of Hyg 
>do you typically use?
>
>Thanks, Aaron.
>Date: Wed, 23 Jun 2004 17:20:14 -0700
>To: tanimoto_h at hotmail.com ("Harumi Tanimoto"), arab-gen at net.bio.net
>From: Zhiyong Wang <zywang24 at stanford.edu>
>Subject: Re: hygromycin resistance
>Cc:
>Bcc:
>X-Attachments:
>
>You will love hygromycin selection if you follow the following 
>procedure. The key is to grow your seedlings in the dark. After you 
>sow the seeds and treat them in cold for a couple of days, put the 
>plates in light for 4-12 hr to promote germination, then put the 
>plates in the dark (wrap up with foil and put in your drawer is good 
>enough). Grow for 5 days (start from the beginning of light 
>treatment, longer growth in the dark will reduce the 
>greening/recovery in light). You will find hyg sensitive seedlings 
>lying on medium with very short hypocotyls and open dotyledons (look 
>like those severe det and cop mutants), and hyg resistant seedlings 
>will be standing up tall like normal dark-grown seedlings (with long 
>hypocotyls and closed cotyledons). Keep the plate in weak light (on 
>you bench) for a day or two to allow hygR seedlings to green up and 
>recover (strong light sometimes bleach the etiolated seedlings, 
>particularly old ones). You can grow them longer in growth chamber 
>to get bigger seedlings and them transfer the tall seedlings to 
>soil. Whatever vector you use, you won't misscore the hygromycin 
>resistant seedlings, unless your transgenic plants have a 
>de-etiolated-in-the-dark phenotype. Have fun.
>
>Zhiyong
>
>Dr. Zhiyong Wang
>Staff Member
>Department of Plant Biology
>Carnegie Institution
>260 Panama street
>Stanford, CA 94305
>
>Phone: 650-325-1521 ext 205
>Fax: 650-325-6857
>><x-flowed>Hi,
>>
>>I am having trouble growing Arabidopsis plants with hygromycin resistance.
>>After selecting on hygromycin and transfering resistant plants to soil, most
>>plants do not survive.  I have spoken to other researchers, some of whom
>>have had the same trouble and some who have not. It seems that it may depend
>>on the vector used and/or the gene being expressed from the vector.
>>
>>Can anybody tell me more specifically what it is that determines how
>>resistant transformants are for  particular vector?  If not, could you let
>>me know whether or not you have had trouble with hygromycin resistance in
>>the past and what vector you used?
>>
>>Thanks,
>>
>>Mimi Tanimoto
>>
>>Department of Plant and Microbial Biology
>>111 Koshland Hall
>>University of California
>>Berkeley
>>CA 94720
>>
>>+1 510 643 9204


--
Dr. Zhiyong Wang
Staff Member
Department of Plant Biology
Carnegie Institution
260 Panama street
Stanford, CA 94305

Phone: 650-325-1521 ext 205
Fax: 650-325-6857




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